Patent Abstract:
Hydrophilic membrane particularly suited for blotting applications, preferably Western blotting. A pre-wet hydrophobic membrane substrate, preferably made of PVDF, is contacted with a monomer solution and subjected to a UV-initiated free radical polymerization step to render the substrate permanently hydrophilic. The resulting membrane exhibits low background fluorescence, high protein binding, excellent retention of protein sample spot morphology, and extended dynamic range (high signal-to-noise ratio, enhanced sample detectability). The membrane demonstrates comparable or higher performance in Western blotting applications than conventional nitrocellulose Western blotting membranes, particularly for protein detection at low sample concentrations, and is directly water-wettable, eliminating the need for an alcohol pre-wet step prior to use.

Full Description:
This application claims priority of Provisional Application Ser. No. 61/189,302 filed Aug. 18, 2008, the disclosure of which is hereby incorporated by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     “Blotting” or “electro-blotting” refers to the process used to transfer biological samples from a gel to a membrane under the influence of an electric field. The process requires a membrane that can immobilize biomolecular samples for subsequent detection. This places specific requirements on the membranes related to surface area, porosity, and protein binding capacity. 
     Western blotting is one modification of this technique that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies. Prior to protein immobilization on the membrane, sample proteins are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) to separate native or denatured proteins. The proteins are then transferred or electro-blotted onto a membrane, where they are probed and ultimately detected using antibodies specific to a target protein. Western blotting membranes are typically made of nitrocellulose (NC) or polyvinylidene fluoride (PVDF). The specificity of the antibody-antigen interaction can enable a single protein to be identified among a complex protein mixture. 
     To summarize, Western blotting involves application of a protein sample (lysate) onto a polyacrylamide gel, subsequent separation of said complex mixture by electrophoresis, and transferal or “electro-blotting” of separated proteins onto a second matrix, generally a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Following the transfer, the membrane is “blocked” to prevent nonspecific binding of antibodies to the membrane surface. Many antibody labeling or tagging strategies are known to those skilled in the art. In the simplest protocols, the transferred proteins are incubated or complexed with a primary enzyme-labeled antibody that serves as a probe. After blocking non-specific binding sites a suitable substrate is added to complex with the enzyme, and together they react to form chromogenic, chemiluminescent, or fluorogenic detectable products that allow for visual, chemiluminescence, or fluorescence detection, respectively. The most sensitive detection schemes make use of chemiluminescent or fluorescent phenomena. In chemiluminescent detection, an enzyme-substrate complex produces detectable optical emissions (chemiluminescence). These emissions are recorded and measured using suitable detectors such as film or photonic devices. Absence or presence of signal indicates whether a specific protein is present in the lysate, and signal intensity is related to the level of the protein of interest, which in some cases may be quantifiable. 
     The use of nitrocellulose membranes is ubiquitous in immunodetection assay work, particularly in Western blotting. This is partially due to historical considerations, and partially due to ease of use. Nitrocellulose blotting membranes do not require an organic liquid pre-wet step, a requirement for working with hydrophobic membranes. Hydrophobic membranes require an alcohol pre-wet step followed by a water exchange step (for alcohol removal), before assembly within the blot-transfer assembly. Intrinsically hydrophobic membranes afford a limited time-frame for this assembly; the potential for the membrane to dry out is significant. Once dry, the membrane cannot be re-wet unless the pre-wet sequence is repeated. Once the membrane is contacted to the gel, removal prior to transfer can effectively ruin the gel and the separated protein samples contained. The pre-wet step is time consuming and can considerably impede workflow. A hydrophilic membrane will remain wet for a longer time interval, and can be re-wet with water if it does dry out before assembly. 
     Nitrocellulose blotting membranes are water wet-able and show satisfactory performance for most blotting applications. But nitrocellulose is not as mechanically or chemically stable as PVDF. PVDF will maintain its mechanical integrity over a long timeframe, whereas NC will become brittle and discolored. PVDF membrane blots can be stripped of antibodies and be re-probed. NC blots cannot. NC is prone to air oxidation, wherein it can become hazardous. It requires a separate waste stream, and when disposed of must be damped with a wetting agent, usually water. 
     Hydrophobic PVDF blotting membranes possess equivalent protein binding ability to NC blotting membranes, but exhibit superior blotting performance. Much lower sample concentrations can be detected under the same conditions on these PVDF membranes compared to NC. Low-background fluorescence hydrophobic PVDF blotting membranes exhibit the same enhanced sample detection while enabling the use of fluorescent detection schemes. 
     It therefore would be desirable to provide a hydrophilic PVDF membrane for immunodetection assays such as Western blotting, with performance characteristics that approach the lower sample detection limits and low background fluorescence that are characteristic of hydrophobic PVDF membranes. This invention addresses these requirements. 
     SUMMARY OF THE INVENTION 
     Those skilled in the art of surface modification for the purpose of altering substrate surface energies, and in particular with regard to surfaces intended for contact with biological systems, will concur that hydrophilic surface modifications traditionally exhibit low protein binding behavior. The embodiments disclosed herein build on the serendipitous and unexpected discovery that space polymers derived from certain monomeric acrylamide mixtures, and formed using free-radical polymerization reactions, can give rise to surface modifications that are not only hydrophilic, but also demonstrate a high level of protein binding. 
     Much of the prior art describes the use of hydroxyl containing monomers, usually carbonyl ester containing acrylate polymers, to produce membrane surface modifications having hydrophilic character and high resistance to protein binding. However, it is known that polymers from such monomers are not resistant to strong alkaline solutions. For example, a solution of 1.0 normal sodium hydroxide will hydrolyze the carbonyl containing acrylate polymers to acrylic acid containing polymers. Such acrylic acid containing polymers are ionically charged under certain pH conditions, and will attract and bind oppositely charged proteins or biomolecules, thus increasing sorption and membrane fouling. In addition, acrylic acid containing polymers swell in water to an extent that they constrict pore passages, thus reducing membrane permeability and productivity. Moreover, polymers from hydroxyl containing monomers, such as hydroxy acrylates, further react in strong alkaline solutions and degrade into soluble low molecular weight fragments, which dissolve away and expose the underlying substrate porous media or membrane. 
     Practitioners attempting to develop optimized membranes either for filtration or non-filtration applications in the pharmaceutical and biotechnology industries must overcome significant problems. Facing stringent cost, performance and safety requirements, a practitioner must use materials and develop manufacturing methods that produce membranes with not only optimized flow and retention characteristics, but be economical to produce, meet cleanliness criteria, be stable to the various chemical environments which are commonly encountered, and be very either very resistant to biomolecule adsorption, or very strongly adsorbing, depending upon the intended end-use. Thus, in this instance, it is very desirable to have a membrane modification that results in a hydrophilic, biomolecule adsorptive surface that is heat stable, which is resistant to degradation by any potential reagent solutions, and which has very low levels of material capable of being extracted there-from. 
     Protein binding results from early investigations into mixed-acrylamide polymeric surface modifications indicated that certain mixtures of hydrophilic bis-acrylamide crosslinking monomers and monofunctional neutral or charged acrylamides can, when copolymerized using UV-initiated or electron beam-initiated free-radical techniques, produce high protein-binding hydrophilic surface modifications. However, initial starting levels of each monomer had to be severely decreased before observing satisfactory dot blot morphology and blotting transfer performance. 
     The problems of the prior art have been overcome by the present embodiments, which provide a hydrophilic membrane particularly suited for blotting applications, preferably Western blotting. More specifically, a pre-wet hydrophobic membrane substrate, preferably made of PVDF, is contacted with a monomer solution and subjected to polymerizing conditions to render the substrate permanently hydrophilic. 
     The resulting membrane exhibits low background fluorescence, high protein binding, excellent retention of protein sample spot morphology, and extended dynamic range (high signal-to-noise ratio, enhanced sample detectability). Where chemiluminescence is used for detection, the level of background fluorescence inherent in the unmodified parent membrane is not as critical. The membrane demonstrates comparable or higher performance in Western blotting applications than conventional nitrocellulose blotting membranes, particularly for detection at low sample concentrations, and is directly water-wettable, eliminating the need for an alcohol pre-wet step prior to use. The membrane exhibits complete, instant, and uniform wetting upon contact with water, and exhibits delayed wetting when contacted with a saturated aqueous aluminum chloride solution. That is, when said membrane is placed on the surface of this saturated aqueous aluminum chloride solution, it wets through in a minimum time interval of not less than 1 second). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1-3  are diagrams of Western blot results using treated membranes in accordance with certain embodiments. 
     
    
    
     DETAILED DESCRIPTION 
     The membranes hydrophilically modified in accordance with embodiments of the present disclosure provide immunodetection assay platforms that are comparable to, or exhibit superior blotting performance to nitrocellulose membranes, particularly with respect to expansion of the low end of the dynamic range of sample detectability. For example, in  FIG. 1 , Western blotting results from a typical development run demonstrate the performance differences between the hydrophilic PVDF blotting membrane of this invention, and the controls (FL—hydrophobic PVDF membrane, and NC—Whatman/S&amp;S BA-85 membrane). Each horizontal strip in the figure contains 5 separate Western transfer blots; three blots on hydrophilic PVDF development samples, and one blot on each of the control membranes. Each horizontal strip of 5 Western blots is the result from one electrophoresis and transfer experiment (5 gels followed by 5 blots were run in each experiment). By design, each experiment embodies identical conditions on each gel/blot with identical quantities of protein sample (applied in 4 lanes across each gel) before electrophoresis and transfer. The results shown are the recorded (chemiluminescent) transfer blots for the detection of 2 proteins (HSP70 and GAPDH) from a complex sample mixture (lysate), applied at decreasing sample concentrations, from left-to-right on each gel, and corresponding to: 5 ug, 2.5 ug, 1.25 ug, 0.67 ug. 
     Suitable porous membranes include those formed from aromatic sulfone polymers, polytetrafluoroethylene, perfluorinated thermoplastic polymers, polyolefin polymers, ultrahigh molecular weight polyethylene, polyamides including Nylon 6 and Nylon 66, and polyvinylidene fluoride, with polyvinylidene fluoride being particularly preferred. Porous membranes include both microporous membranes and ultrafiltration membranes, and are preferably in the form of sheets. Generally the average pore sizes include those between 0.001 and 10 microns. Blotting membranes are nominally 0.45 um pore size materials. Preferred starting membranes have a porosity (void volume) range specification of 68-73%. Blotting membranes are traditionally symmetric. However, the coating could be applied to an asymmetric membrane. 
     The polymeric coating can be a copolymer or terpolymer formed from at least one polyfunctional monomer modified with at least one hydrophilic functional group, said hydrophilic polyfunctional monomer(s) selected from the group consisting of polyfunctional acrylamides, polyfunctional methacrylamides and diacroylpiperazines, and formed from at least one monofunctional monomer modified with at least one hydrophilic functional group, said hydrophilic monofunctional monomer(s) selected from the group consisting of monofunctional acrylamides, monofunctional methacrylamides, and acryloyl piperazines. 
     It was found that a porous hydrophobic membrane, preferably one made of polyvinylidene fluoride coated with a crosslinked acrylamide-methylene-bis-acrylamide copolymer was rendered highly hydrophilic. Furthermore, at the copolymer level that was applied to the porous PVDF membrane samples in early iterations of this invention, IgG binding assays revealed protein binding levels to be in the neighborhood of 400 ug/cm 2 . This level is typical of the parent hydrophobic PVDF membrane and of conventional nitrocellulose membranes. The first surprising result was that membranes so prepared were both hydrophilic, and high protein-binding. However, the Western blotting performance of these initial samples (those that exhibited this high level of protein binding) was not satisfactory in terms of maintaining small sample blot size (blot morphology), and in terms of sample capture in blot transfers. By modifying the copolymer coating level, satisfactory blotting performance was realized. At these modified levels, protein binding levels were reduced to between 250 and 325(00) ug/cm 2 , but Western blotting performance rose to levels intermediate between nitrocellulose and the preferred parent hydrophobic PVDF membrane. Surprisingly, the present inventors found that the protein binding level is not the best or only predictor of Western blotting membrane performance. Sacrificing some protein binding ability by modifying the coating level on the substrate can result in improved blotting performance. 
     Thus, component levels and relative concentrations of the modifying formulation are critical to obtain acceptable immunodetection assay performance. A low overall solids concentration in a highly specific component ratio balances water-wetting performance against blotting performance. The very low background fluorescence level of the substrate membrane is preserved. If, however, the level of surface modification chemistry is too low, the result is a membrane that is not water-wettable to an acceptable extent. If the level of surface modification is too high the resulting membranes exhibit extremely high surface energies. As stated earlier, at higher levels of surface modifying chemistry, the measured protein binding capacity is roughly equivalent to nitrocellulose and hydrophobic PVDF membranes, but poor electro-blotting performance results. 
     In accordance with certain embodiments, the total solids level in the modifying/reactant solution is to be adjusted to between 0.90% and 1.10% by weight. A total solids concentration in this range with the specified component ratio results in optimal blotting performance. This formulation includes a UV-photoinitiator component. 
     Suitable amounts of the acrylamide monofunctional monomer and the bis-acrylamide crosslinking monomer in the reactant solution are to be between 0.20% and 2.00% by weight (each), preferably with amounts between 0.30% and 0.60% by weight (each), and most preferably between 0.40% and 0.50% by weight (inclusive, each). The preferred ratio of acrylamide to bis-acrylamide of the monomer reactant solution is about 1:1 (mass/mass). The preferred overall monomer concentration of acrylamide:methylene-bis-acrylamide monomer reactant solution is between 0.5% and 1.5% by mass. A suitable UV-photoinitiator component is present in 0.01% to 0.20% by weight preferably between 0.05 and 0.15% by weight, and most preferably between 0.09% and 0.11%, by weight. Suitable UV-photoinitiators include IRGACURE® 2959 (2-hydroxyl-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Ciba-geigy) 500, 754, 2959, and 819DW. Methods for preparation of the modified porous membrane substrate in accordance with certain embodiments include the steps of providing a porous membrane substrate, contacting the surface of the porous membrane substrate with a reactant solution comprising acrylamide and methylene-bis-acrylamide, and a suitable photoinitiator, removing the membrane from the solution, and polymerizing the coating in situ on the membrane substrate by exposing the same to radiation of a suitable wavelength and intensity for a suitable time interval. Preferably the porous membrane contacted with the reaction solution is irradiated with an ultraviolet light source. Filters may be used to reduce or eliminate undesirable wavelengths which may cause damage to the porous membrane. The amount of exposure time to the UV light and the intensity thereof should be familiar to those skilled in the art. 
     In the preferred embodiment of the invention, a laboratory-scale preparation of the reactant monomer solution is made by dissolving 1.00 g acrylamide, [H 2 C═CH—C(O)—NH 2 ] monofunctional monomer; 0.80 g methylene-bis-acrylamide, [H 2 C(—NH—C(O)CH═CH 2 ) 2 ] cross-linking monomer; and, 0.20 g Irgacure 2959 photo-initiator into 198.00 g of Milli-Q® water. An extended mixing interval of about 2 hours is required to fully dissolve the cross-linker and the photo-initiator. 
     More specifically, the porous hydrophobic starting membrane is pre-wet by immersion in an organic liquid or in an aqueous solution thereof that does not swell or dissolve the porous membrane, and which pre-wets the entire porous surface of the membrane. 
     The liquid may be a low molecular weight alcohol, or a mixture of water and a miscible organic liquid. Suitable liquids or compositions include methanol, ethanol, isopropanol, water mixtures thereof, acetone/water mixtures, and tetrahydrofuran/water mixtures of sufficiently low surface tension to affect wetting the entire membrane surface. 
     The purpose of this pre-wetting step is to assure that the entire membrane surface is rendered wettable by water, and subsequently by the aqueous reactant monomer solution. The pre-wetting step must be followed by a rigorous exchange step with water to eliminate the presence of the organic solvent. These pre-wetting solvents or water mixtures thereof can exert a negative influence upon the intended polymerization of the reactant monomers. 
     Subsequent immersion and gentle agitation of the water-wet porous membrane in the reactant solution allows the entire surface of the porous membrane to be wet with reactant solution. As long as excess water is removed from the membrane prior to immersion in the reactant solution, no significant dilution of the reactant solution will occur. 
     The sample is withdrawn after a short (two minute) time interval and excess reactant solution is removed from the membrane sample. The reactant solution-wetted membrane is anaerobically exposed to UV radiation to effect the polymerization directly onto the entire porous membrane surface. The resulting coated membrane exhibits: Immediate, complete, and thoroughly uniform wetting when contacted onto a water surface; A high level of Western blotting performance; A high level of protein binding (≧250 ug/cm 2  IgG) by radio-labeled assay; and, Low background fluorescence (about 2000 rfu @ 485 nm/535 nm excitation/emission wavelengths using a TECAN GENios FL fluorescence reader with detector gain set at 86, and running Magellan 5.0 software package), which is about twice the background fluorescence of the untreated (unmodified parent hydrophobic) membrane under the same measurement conditions. When placed on the surface of a saturated aqueous aluminum chloride solution, the membrane will wet through in a minimum time interval of not less than 1 second, and in a maximum time interval that may exceed 60 seconds. 
     EXAMPLE 1 
     Commercially available hydrophobic PVDF membrane from Millipore Corporation (IPFL00000) was immersed in methyl alcohol. The membrane was withdrawn and immersed in water with agitation to extract methanol for 1 minute. The membrane was withdrawn and immersed in fresh water for an additional 2-minute interval and then placed in fresh water before immersion in reactant monomer solution. Excess water was drained from the membrane and the membrane was then immersed in monomer reactant solution with gentle agitation for 2 minutes. Membrane was then exposed to UV radiation from both sides in a UV curing process at a line speed of 15 to 25 fpm. Membrane was recovered and placed into a water bath to remove unreacted monomer and non-adhering oligomers and polymer. Samples were dried either in air at room temperature overnight, or in a static forced-air oven between 60° C. and 80° C. for 10 minutes, or on an impingement dryer at 90-110° C. at a line speed of 15 to 25 fpm. Average membrane extractables as determined by an in-house TOC (Total Organic Carbon) method were measured to be about 1.44 ug/cm2, as shown in Table 1. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Total Organic Carbon (TOC) - Five (5) 47 mm disks. 
               
               
                 Requestor ID: ML6UJP37 
               
               
                 Requestor: Antoni Peters 
               
               
                 PVDF Hydrophillic IPFL Membrane Received 
               
               
                 For Blotting and Immuno Assays Applications 
               
               
                 Ext. for TOC Analysis 
               
               
                 Extraction By: A. Pervez 
               
               
                 TOC By: M. Santos-Rosa &amp; A. Pervez 
               
               
                 Date: Oct. 16-19, 2006 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Ext. Temp. 
                 Ext. Time 
                 Ext. Vol. 
                 Ext. Area 
               
               
                 Ext. Solvent 
                 [° C.] 
                 [Hours] 
                 [g] 
                 [cm 2] 
               
               
                   
               
               
                 MilliQ Water 
                 Ambient 
                 24 
                 40 
                 86.75 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 Summary 
               
               
                   
                   
                   
                   
                   
                 Results 
               
               
                   
                 TOC Acid/Oxid 
                 TOC 
                 Corrected TOC 
                 TOC 
                 TOC 
               
               
                 Sample ID 
                 [μL/min] 
                 [ppm C] 
                 [ppm C] 
                 [μg C] 
                 [μg C/cm 2] 
               
               
                   
               
               
                 Water Blank 
                 0.20/0.20 
                 0.0639 
                   
                   
                   
               
               
                 T102 072806 A: -MBAM/AC/I-2959 
                 0.75/1.00 
                 3.29 
                 3.23 
                 129 
                 1.49 
               
               
                 T102 072806 B: -MBAM/AC/I-2959 
                 0.75/1.00 
                 2.91 
                 2.85 
                 114 
                 1.31 
               
               
                 T102 072806 C: -MBAM/AC/I-2959 
                 0.75/1.00 
                 3.33 
                 3.27 
                 131 
                 1.51 
               
               
                   
               
             
          
         
       
     
     Average extractable residual monomer levels were determined by HPLC. Values determined from 3 samples are provided in Table 2. 
     
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Extractable Monomer Levels by HPLC - 
               
               
                 Same samples as shown in Table 1. 
               
             
          
           
               
                   
                 Acrylamide 
                 MBAM 
                 Irgacure(R) 2959 
               
               
                 Sample 
                 (μg/cm2) 
                 (μg/cm2) 
                 (μg/cm2) 
               
               
                   
               
               
                 T102 072806A 
                 0.002 
                 0.105 
                 N.D. 
               
               
                 T102 072806B 
                 0.005 
                 0.091 
                 N.D. 
               
               
                 T102 072806C 
                 0.011 
                 0.094 
                 N.D. 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 2 
     The procedure of Example 1 was used to treat PVDF membranes having the specifications, treatment conditions and reactant solutions shown in Tables 3A-D, 4A-D, and 5A-D. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3A 
               
             
             
               
                   
               
               
                 MODIFICATION DATA for Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                   
                   
                 Membrane 
                   
               
               
                   
                 Monomer 
                 Starting 
                 Casting 
                 Starting Membrane Properties 
               
             
          
           
               
                   
                 Mix 
                 Membrane 
                 Dryer 
                   
                   
                   
                 Flow Time 
               
               
                 Roll No. 
                 Lot# 
                 Lot Data 
                 Temperature (F.) 
                 Thick (um) 
                 Porosity (%) 
                 Bbl Pt (psi) 
                 (seconds) 
               
               
                   
               
             
          
           
               
                 RUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 R01 
                 R Mix 1 
                 IPFL 071007 T214 
                 300 
                 112 
                 72.8 
                 9.4 
                 56.0 
               
               
                 R02 
                 R Mix 1 
                 IPFL 071007 T205 
                 300 
                 113 
                 73.0 
                 9.4 
                 63.0 
               
               
                 R03 
                 R Mix 1 
                 IPFL 071007 T205 
                 300 
                 113 
                 73.0 
                 9.4 
                 63.0 
               
               
                 R04 
                 R Mix 1 
                 IPFL 071007 T205 
                 300 
                 113 
                 73.0 
                 9.4 
                 63.0 
               
               
                 R05 
                 R Mix 1 
                 IPFL 071007 T205 
                 300 
                 113 
                 73.0 
                 9.4 
                 63.0 
               
               
                 R06 
                 R Mix 1 
                 IPFL 071007 T206 
                 300 
                 111 
                 72.4 
                 9.5 
                 65.0 
               
               
                 R07 
                 R Mix 1 
                 IPFL 071007 T206 
                 300 
                 111 
                 72.4 
                 9.5 
                 65.0 
               
               
                 R08 
                 R Mix 1 
                 IPFL 071007 T206 
                 300 
                 111 
                 72.4 
                 9.5 
                 65.0 
               
               
                 R09 
                 R Mix 1 
                 IPFL 071007 T206 
                 300 
                 111 
                 72.4 
                 9.5 
                 65.0 
               
               
                 R10 
                 R Mix 1 
                 IPFL 071007 T214 
                 300 
                 112 
                 72.8 
                 9.4 
                 56.0 
               
               
                 R11 
                 R Mix 1 
                 IPFL 071007 T214 
                 300 
                 112 
                 72.8 
                 9.4 
                 56.0 
               
               
                 R12 
                 R Mix 1 
                 IPFL 071007 T214 
                 300 
                 112 
                 72.8 
                 9.4 
                 56.0 
               
               
                 R12 End 
                 NA 
                 NA 
                 300 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3B 
               
             
             
               
                   
               
               
                 MODIFICATION DATA for Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Monomer Mix Component Concentrations 
                   
               
             
          
           
               
                   
                   
                 Actual 
                 Actual 
                 Actual 
                 Actual 
                 Totalized 
               
               
                   
                 Monomer Mix 
                 AC 
                 MBAM 
                 I-2959 
                 Total Solids 
                 Footage 
               
               
                 Roll No. 
                 Sample ID 
                 Wt % (HPLC) 
                 Wt % (HPLC) 
                 Wt % (HPLC) 
                 Wt % (HPLC) 
                 By Roll 
               
               
                   
               
             
          
           
               
                 RUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration 
               
             
          
           
               
                 NA 
                 R Mix 1 DRUM 
                 0.5156 
                 0.4214 
                 0.1130 
                 1.0500 
                 0 
               
               
                 R01 
                 MM1 Start 
                 0.5091 
                 0.4042 
                 0.1102 
                 1.0235 
                 0 
               
               
                 R02 
                 MM2 
                 0.5105 
                 0.3957 
                 0.1094 
                 1.0156 
                 500 
               
               
                 R03 
                 MM3 
                 0.5038 
                 0.3837 
                 0.1055 
                 0.9931 
                 800 
               
               
                 R04 
                 MM4 
                 0.4996 
                 0.3773 
                 0.1001 
                 0.9771 
                 1100 
               
               
                 R05 
                 MM5 
                 0.4948 
                 0.3708 
                 0.0999 
                 0.9655 
                 1250 
               
               
                 R06 
                 MM6 
                 0.4935 
                 0.3685 
                 0.1020 
                 0.9640 
                 1550 
               
               
                 R07 
                 MM7 
                 0.4868 
                 0.3618 
                 0.0995 
                 0.9481 
                 1850 
               
               
                 R08 
                 MM8 
                 0.4868 
                 0.3605 
                 0.0974 
                 0.9448 
                 2150 
               
               
                 R09 
                 MM9 
                 0.4777 
                 0.3553 
                 0.0956 
                 0.9286 
                 2300 
               
               
                 R10 
                 MM10 
                 0.4721 
                 0.3528 
                 0.0956 
                 0.9204 
                 2600 
               
               
                 R11 
                 MM11 
                 0.4673 
                 0.3521 
                 0.0932 
                 0.9125 
                 2900 
               
               
                 R12 
                 MM12 
                 0.4682 
                 0.3561 
                 0.0918 
                 0.9161 
                 3200 
               
               
                 R12 End 
                 MM13 End 
                 0.4574 
                 0.3546 
                 0.0879 
                 0.8999 
                 3300 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3C 
               
             
             
               
                   
               
               
                 MODIFICATION DATA for Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Nip 
                 Nip 
                   
                 UV Chamber Conditions 
                   
               
             
          
           
               
                   
                 Aisle 
                 Wall 
                 Line 
                   
                   
                 N 2  Flow 
                   
                   
                   
                 Starting 
               
               
                   
                 Press 
                 Press 
                 Speed 
                 Lamps 
                 Lamps 
                 Top/Bottom 
                 UV P1 
                 UV P3 
                 UV P2 
                 O 2  Level 
               
               
                 Roll No. 
                 psi 
                 psi 
                 ft/min 
                 Type/No 
                 Config 
                 (SCFM) 
                 Inch HWC 
                 Inch HWC 
                 Inch HWC 
                 (ppm) 
               
               
                   
               
             
          
           
               
                 RUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 1.0 
                 1.6 
                 1.0 
                 90.0 
               
               
                 R01 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 70.0 
               
               
                 R02 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 56.7 
               
               
                 R03 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 39.3 
               
               
                 R04 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 33.0 
               
               
                 R05 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 28.8 
               
               
                 R06 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 27.8 
               
               
                 R07 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 26.9 
               
               
                 R08 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 26.4 
               
               
                 R09 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 27.4 
               
               
                 R10 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 25.2 
               
               
                 R11 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 27.1 
               
               
                 R12 
                 20 
                 20 
                 20 
                 H/4 
                 Staggered 
                 10/9.5 
                 1.1 
                 1.7 
                 1.1 
                 27.8 
               
               
                 R12 End 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 1.1 
                 1.7 
                 1.1 
                 25.2 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3D 
               
             
             
               
                   
               
               
                 MODIFICATION DATA for Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Water Wet 
                 Water Wet 
                 Water Wet 
                 Salt Wet 
                 Salt Wet 
                 Salt Wet 
                 Salt Wet 
                 Fluores 
                 QC Blotting 
                 Western 
               
               
                   
                 Speed 
                 Uniform 
                 Through 
                 Time 
                 Time 
                 Time 
                 Time 
                 BKG 
                 Pass/Fail 
                 Blotting 
               
               
                 Roll No. 
                 OK? 
                 X-web? 
                 OK? 
                 Seconds 
                 Seconds 
                 Seconds 
                 Seconds 
                 RFU 
                 Control 
                 Live Lysate 
               
               
                   
               
             
          
           
               
                 RUN R - No Mix Ajustment - Footage VS Monomer Mix Concentration 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 ≧NC 
               
               
                 R01 
                 Y 
                 Y 
                 Y 
                 4.7 
                 3.5 
                 3.0 
                 3.7 
                 1750.57 
                 Pass 9 Band 
                 ≧NC 
               
               
                 R02 
                 Y 
                 Y 
                 Y 
                 1.4 
                 2.4 
                 2.4 
                 2.1 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R03 
                 Y 
                 Y 
                 Y 
                 1.4 
                 2.4 
                 2.4 
                 2.1 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R04 
                 Y 
                 Y 
                 Y 
                 2.1 
                 2.2 
                 2.5 
                 2.3 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R05 
                 Y 
                 Y 
                 Y 
                 2.9 
                 3.1 
                 2.9 
                 3.0 
                 2015.63 
                 Pass 9 Band 
                 ≧NC 
               
               
                 R06 
                 Y 
                 Y 
                 Y 
                 2.2 
                 2.3 
                 2.3 
                 2.3 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R07 
                 Y 
                 Y 
                 Y 
                 4.1 
                 3.9 
                 4.0 
                 4.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R08 
                 Y 
                 Y 
                 Y 
                 2.3 
                 3.7 
                 3.2 
                 3.1 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R09 
                 Y 
                 Y 
                 Y 
                 5.7 
                 7.3 
                 5.3 
                 6.1 
                 1927.95 
                 Pass 9 Band 
                 ≧NC 
               
               
                 R10 
                 Y 
                 Y 
                 Y 
                 5.4 
                 7.1 
                 5.4 
                 6.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R11 
                 Y 
                 Y 
                 Y 
                 6.3 
                 7.5 
                 5.6 
                 6.5 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 R12 
                 Y 
                 Y 
                 Y 
                 8.5 
                 7.8 
                 7.6 
                 8.0 
                 1708.5  
                 Pass 9 Band 
                 ≧NC 
               
               
                 R12 End 
                 Y 
                 Y 
                 Y 
                 16.7 
                 16.5 
                 16.3 
                 16.5 
                 NA 
                 NA 
                 ≧NC 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 4A 
               
             
             
               
                   
               
               
                 MEMBRANE MODIFICATION DATA - Hydrophilic 
               
               
                 PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                   
                   
                   
                 Heat Treat 
                   
               
               
                   
                   
                   
                   
                 Temperature 
                 Membrane 
               
               
                   
                   
                 Monomer 
                 Starting 
                 or Mod Line 
                 Casting 
               
               
                 Run 
                   
                 Mix 
                 Membrane 
                 Dryer 
                 Dryer 
               
               
                 Segment 
                 Roll No. 
                 Lot# 
                 Lot Data 
                 Temperature 
                 Temperature 
               
               
                   
               
             
          
           
               
                 RUN S - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                   
                   
               
               
                 Test 
                 R01 
                 S Mix 1 
                 IPFL 071007 T203 
                 205 
                 300 
               
               
                 S1 
                 R02 
                 S Mix 1 
                 IPX 070907 R103 
                 205 
                 300 
               
               
                 S1 
                 R03 
                 S Mix 1 
                 IPX 091407 T103 
                 205 
                 310 
               
               
                 S1 
                 R04 
                 S Mix 1 
                 IPX 120307 T107 
                 205 
                 300 
               
               
                 S1 
                 R05 
                 S Mix 1 
                 IPX 091407 T102 
                 205 
                 310 
               
               
                 S1 
                 R06 
                 S Mix 1 
                 IPX 070907 R105 
                 204 
                 300 
               
               
                 S2 
                 R07 
                 S Mix 1 
                 IPX 120307 T107 
                 200 
                 300 
               
               
                 S2 
                 R08 
                 S Mix 1 
                 IPX 120307 T107 
                 Transition 
                 300 
               
               
                 S2 
                 R09 
                 S Mix 1 
                 IPX 120307 T107 
                 205 
                 300 
               
               
                 S2 
                 R10 
                 S Mix 1 
                 IPX 120307 T107 
                 Transition 
                 300 
               
               
                 S2 
                 R11 
                 S Mix 1 
                 IPX 120307 T107 
                 210 
                 300 
               
               
                 S2 
                 R12 
                 S Mix 1 
                 IPX 120307 T107 
                 210 
                 300 
               
               
                 S2 
                 R13 
                 S Mix 1 
                 IPX 120307 T107 
                 210 
                 300 
               
               
                   
               
               
                 Segment 1 Variation of starting membrane properties 
               
               
                 Segment 2 Variation of line speed and dryer temperature 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4B 
               
             
             
               
                   
               
               
                 MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Monomer Mix Samples and Mix Component Concentrations 
                   
               
             
          
           
               
                   
                 Monomer 
                 Actual 
                 Actual 
                 Actual 
                 Actual 
                   
               
             
          
           
               
                   
                 Starting Membrane Properties 
                 Mix 
                 AC 
                 MBAM 
                 I-2959 
                 Total Solids 
                 Totalized 
               
             
          
           
               
                 Run 
                 Roll 
                 Thick 
                 Porosity 
                 Bbl Pt 
                 Flow Time 
                 Sample 
                 Wt % 
                 Wt % 
                 Wt % 
                 Wt % 
                 Footage 
               
               
                 Segment 
                 No. 
                 (um) 
                 (%) 
                 (psi) 
                 (seconds) 
                 ID 
                 (HPLC) 
                 (HPLC) 
                 (HPLC) 
                 (HPLC) 
                 By Roll 
               
               
                   
               
             
          
           
               
                 RUN S - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 MM1B 
                 0.4979 
                 0.3894 
                 0.1005 
                 0.9878 
                 150 
               
               
                 Test 
                 R01 
                 112 
                 72.8 
                 9.6 
                 61.0 
                 MM1C start 
                 0.4926 
                 0.3877 
                 0.1005 
                 0.9808 
                 300 
               
               
                 S1 
                 R02 
                 115 
                 74.8 
                 9.3 
                 55.0 
                 MM2B 
                 0.4893 
                 0.3849 
                 0.0996 
                 0.9738 
                 450 
               
               
                 S1 
                 R03 
                 115 
                 66.4 
                 9.6 
                 59.0 
                 MM3B 
                 0.4812 
                 0.3785 
                 0.0971 
                 0.9568 
                 600 
               
               
                 S1 
                 R04 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM4B 
                 0.4780 
                 0.3760 
                 0.0963 
                 0.9503 
                 750 
               
               
                 S1 
                 R05 
                 122 
                 66.2 
                 11.1 
                 78.3 
                 MM5B 
                 0.4761 
                 0.3733 
                 0.0954 
                 0.9448 
                 950 
               
               
                 S1 
                 R06 
                 130 
                 74.7 
                 10.7 
                 74.0 
                 MM6B 
                 0.4785 
                 0.3757 
                 0.0953 
                 0.9495 
                 1100 
               
               
                 S2 
                 R07 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM7B 
                 0.4699 
                 0.3678 
                 0.0934 
                 0.9311 
                 1200 
               
               
                 S2 
                 R08 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM8B 
                 0.4673 
                 0.3656 
                 0.0926 
                 0.9255 
                 1305 
               
               
                 S2 
                 R09 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM9B 
                 0.4660 
                 0.3644 
                 0.0925 
                 0.9229 
                 1405 
               
               
                 S2 
                 R10 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM10B 
                 0.4655 
                 0.3637 
                 0.0917 
                 0.9209 
                 1505 
               
               
                 S2 
                 R11 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM11B 
                 0.4642 
                 0.3619 
                 0.0910 
                 0.9171 
                 1605 
               
               
                 S2 
                 R12 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM12B 
                 0.4618 
                 0.3598 
                 0.0903 
                 0.9119 
                 1705 
               
               
                 S2 
                 R13 
                 126 
                 72.9 
                 9.4 
                 65.0 
                 MM13B 
                 Not 
                 Not 
                 Not 
                 Not 
                 1855 
               
               
                   
                   
                   
                   
                   
                   
                   
                 Available 
                 Available 
                 Available 
                 Available 
               
               
                   
               
               
                 Segment 1 Variation of starting membrane properties 
               
               
                 Segment 2 Variation of line speed and dryer temperature 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4C 
               
             
             
               
                   
               
               
                 MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Nip 
                 Nip 
                   
                 UV Chamber Conditions 
                   
               
             
          
           
               
                   
                   
                 Aisle 
                 Wall 
                 Line 
                 N 2  Flow 
                   
                   
                   
                 Starting 
               
               
                 Run 
                   
                 Press 
                 Press 
                 Speed 
                 Top/Bottom 
                 UV P1 
                 UV P3 
                 UV P2 
                 O 2  Level 
               
               
                 Segment 
                 Roll No. 
                 psi 
                 psi 
                 ft/min 
                 (SCFM) 
                 Inch HWC 
                 Inch HWC 
                 Inch HWC 
                 (ppm) 
               
               
                   
               
             
          
           
               
                 RUN S - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Test 
                 R01 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 15.0 
               
               
                 S1 
                 R02 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 42.0 
               
               
                 S1 
                 R03 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 40.0 
               
               
                 S1 
                 R04 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 40.0 
               
               
                 S1 
                 R05 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 40.0 
               
               
                 S1 
                 R06 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.5 
                 1.1 
                 40.0 
               
               
                 S2 
                 R07 
                 20 
                 20 
                 22 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 53.7 
               
               
                 S2 
                 R08 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 39.1 
               
               
                 S2 
                 R09 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 37.6 
               
               
                 S2 
                 R10 
                 20 
                 20 
                 18 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 33.0 
               
               
                 S2 
                 R11 
                 20 
                 20 
                 18 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 30.5 
               
               
                 S2 
                 R12 
                 20 
                 20 
                 20 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 24.9 
               
               
                 S2 
                 R13 
                 20 
                 20 
                 25 
                 10/9.5 
                 1.0 
                 1.4 
                 1.1 
                 25.2 
               
               
                   
               
               
                 Segment 1 Variation of starting membrane properties 
               
               
                 Segment 2 Variation of line speed and dryer temperature 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4D 
               
             
             
               
                   
               
               
                 MEMBRANE MODIFICATION DATA - Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                   
                 Water Wet 
                 Water Wet 
                 Water Wet 
                 Salt Wet 
                 Salt Wet 
                 Salt Wet 
                 Salt Wet 
                 Fluores 
                 QC Blotting 
                 Western 
               
               
                 Run 
                 Roll 
                 Speed 
                 Uniform 
                 Through 
                 Time 
                 Time 
                 Time 
                 Time 
                 BKG 
                 Pass/Fail 
                 Blotting 
               
               
                 Segment 
                 No. 
                 OK? 
                 X-web? 
                 OK? 
                 Seconds 
                 Seconds 
                 Seconds 
                 Seconds 
                 RFU 
                 Control 
                 Live Lysate 
               
               
                   
               
             
          
           
               
                 RUN S - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 Test 
                 R01 
                 Y 
                 Y 
                 Y 
                       8.2 
                 8.8 
                 4.9 
                 7.3 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S1 
                 R02 
                 Y 
                 Y 
                 Y 
                      26.2 
                 15.2 
                 5.3 
                 15.6 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S1 
                 R03 
                 Y 
                 Y 
                 Y 
                       4.2 
                 4.2 
                 2.9 
                 3.8 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S1 
                 R04 
                 Y 
                 Y 
                 Y 
                       5.4 
                 13.6 
                 10.3 
                 9.8 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S1 
                 R05 
                 Y 
                 N 
                 N 
                       7.8 
                 4.6 
                 5.7 
                 6.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S1 
                 R06 
                 Y 
                 Y 
                 Y 
                      12.7 
                 8.0 
                 9.9 
                 10.2 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R07 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 90.0 
                 25.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R08 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 &gt;120 
                 40.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R09 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 &gt;120 
                 30.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R10 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 &gt;120 
                 55.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R11 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 &gt;120 
                 80.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R12 
                 Y 
                 Y 
                 Y 
                 &gt;120* 
                 &gt;120 
                 80.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 S2 
                 R13 
                 Y 
                 Y 
                 Y 
                 &gt;120  
                 &gt;120 
                 55.0 
                 Meaningless 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                   
               
               
                 Segment 1 Variation of starting membrane properties 
               
               
                 Segment 2 Variation of line speed and dryer temperature 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5A 
               
             
             
               
                   
               
               
                 MODIFICATION DATA FOR Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                   
                   
                   
                   
                 Heat Treat 
                   
               
               
                   
                   
                   
                   
                   
                 Temperature 
                 Membrane 
               
               
                   
                   
                 Monomer 
                 Starting 
                   
                 or Mod Line 
                 Casting 
               
               
                 Run 
                   
                 Mix 
                 Membrane 
                 Good 
                 Dryer 
                 Dryer 
               
               
                 Segment 
                 Roll No. 
                 Lot# 
                 Lot Data 
                 Footage 
                 Temperature 
                 Temperature 
               
               
                   
               
             
          
           
               
                 RUN T - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 1 
                 NA 
                 010908M1DRUM 
                 0 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 010908M1-MM1 Tank 
                 0 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 010908M1-MM2 Tank Diluted 
                 0 
                 NA 
                 NA 
               
             
          
           
               
                 1 
                 R01 
                 010908M1 
                 IPVH 050107 T101B 
                 155 
                 200 
                   
               
               
                 1 
                 R02 
                 010908M1 
                 IPFL 071007 T203 
                 90 
                 200 
                 300 
               
               
                 2 
                 R03 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 200 
                 300 
               
               
                 2 
                 R04 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 200 
                 300 
               
               
                 2 
                 R05 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 Transition 
                 300 
               
               
                 2 
                 R06 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 220 
                 300 
               
               
                 2 
                 R07 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 215 
                 300 
               
               
                 2 
                 R08 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 215 
                 300 
               
               
                 2 
                 R09 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 Transition 
                 300 
               
               
                 2 
                 R10 
                 010908M1 
                 IPX 120307 T109 
                 90 
                 220 
                 300 
               
               
                 3 
                 R11 
                 010908M1 
                 IPX 091407 T101 
                 10 
                 205.4 
                 300 
               
               
                 3 
                 R12 
                 010908M1 
                 IPX 071007 T213 
                 90 
                 205.5 
                 300 
               
               
                 3 
                 R13 
                 010908M1 
                 IPX 120307 T104 
                 90 
                 205.8 
                 300 
               
               
                 3 
                 R14 
                 010908M1 
                 IPX 070907 T105 
                 90 
                 206.1 
                 300 
               
               
                 3 
                 R15 
                 010908M1 
                 IPX 070907 R102 
                 90 
                 205 
                 300 
               
               
                 3 
                 R16 
                 010908M1 
                 IPX 070907 R106 
                 90 
                 205 
                 300 
               
               
                 3 
                 R17 
                 010908M1 
                 IPX 120307 R110 
                 90 
                 205 
                 300 
               
               
                 3 
                 NA 
                 010908M1 
                 NA 
                 0 
                 205 
               
               
                   
               
               
                 Segment 1 Diagnostic 
               
               
                 Segment 2 Dryer Temperature &amp; Linespeed Variation 
               
               
                 Segment 3 Porosity and Thickness Variation - Starting Membrane 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5B 
               
             
             
               
                   
               
               
                 MODIFICATION DATA FOR Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Monomer Mix Samples and Component Concentrations 
                 Actual 
                   
               
             
          
           
               
                   
                 Monomer 
                 Actual 
                 Actual 
                 Actual 
                 Total 
                   
               
             
          
           
               
                   
                 Starting Membrane Properties 
                 Mix 
                 AC 
                 MBAM 
                 I-2959 
                 Solids 
                 Totalized 
               
             
          
           
               
                 Run 
                 Roll 
                 Thick 
                 Porosity 
                 Bbl Pt 
                 Flow Time 
                 Sample 
                 Wt % 
                 Wt % 
                 Wt % 
                 Wt % 
                 Footage 
               
               
                 Segment 
                 No. 
                 (um) 
                 (%) 
                 (psi) 
                 (seconds) 
                 ID 
                 (HPLC) 
                 (HPLC) 
                 (HPLC) 
                 (HPLC) 
                 By Roll 
               
               
                   
               
             
          
           
               
                 RUN T - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 DRUM 
                 0.76 
                 0.60 
                 0.15 
                 1.51 
                 0 
               
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 MM1 
                 0.75 
                 0.58 
                 0.15 
                 1.48 
                 0 
               
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 MM2 Start 
                 0.51 
                 0.40 
                 0.10 
                 1.00 
                 0 
               
               
                 1 
                 R01 
                 109-125 
                 Not 
                 Not 
                 Not 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 375 
               
               
                   
                   
                   
                 Available 
                 Available 
                 Available 
                   
                   
                   
                   
                   
                   
               
               
                 1 
                 R02 
                 112 
                 72.8 
                 9.6 
                 61.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 675 
               
               
                 2 
                 R03 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 MM3 
                 0.50 
                 0.39 
                 0.10 
                 0.99 
                 775 
               
               
                 2 
                 R04 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 875 
               
               
                 2 
                 R05 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 975 
               
               
                 2 
                 R06 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1075 
               
               
                 2 
                 R07 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1175 
               
               
                 2 
                 R08 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1275 
               
               
                 2 
                 R09 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1375 
               
               
                 2 
                 R10 
                 120 
                 71.0 
                 9.5 
                 63.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1675 
               
               
                 3 
                 R11 
                 118 
                 66.7 
                 8.3 
                 46.7 
                 MM4 
                 0.48 
                 0.38 
                 0.09 
                 0.96 
                 1775 
               
               
                 3 
                 R12 
                 116 
                 72.3 
                 9.0 
                 56.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1875 
               
               
                 3 
                 R13 
                 119 
                 72.4 
                 9.7 
                 66.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 1975 
               
               
                 3 
                 R14 
                 130 
                 74.7 
                 10.7 
                 74.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 2075 
               
               
                 3 
                 R15 
                 134 
                 73.4 
                 9.9 
                 65.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 2175 
               
               
                 3 
                 R16 
                 114 
                 72.4 
                 9.7 
                 67.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 2275 
               
               
                 3 
                 R17 
                 113 
                 70.2 
                 9.4 
                 61.0 
                 Not Taken 
                 NA 
                 NA 
                 NA 
                 NA 
                 2375 
               
               
                 3 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 MM5 
                 0.47 
                 0.37 
                 0.09 
                 0.94 
                 2675 
               
               
                   
               
               
                 Segment 1 Diagnostic 
               
               
                 Segment 2 Dryer Temperature &amp; Linespeed Variation 
               
               
                 Segment 3 Porosity and Thickness Variation - Starting Membrane 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5C 
               
             
             
               
                   
               
               
                 MODIFICATION DATA FOR Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                 Nip 
                 Nip 
                   
                 UV Chamber Conditions 
                   
               
             
          
           
               
                   
                   
                 Aisle 
                 Wall 
                 Line 
                 N 2  Flow 
                   
                   
                   
                 Starting 
               
               
                 Run 
                   
                 Press 
                 Press 
                 Speed 
                 Top/Bottom 
                 UV P1 
                 UV P3 
                 UV P2 
                 O 2  Level 
               
               
                 Segment 
                 Roll No. 
                 psi 
                 psi 
                 ft/min 
                 (SCFM) 
                 Inch HWC 
                 Inch HWC 
                 Inch HWC 
                 (ppm) 
               
               
                   
               
             
          
           
               
                 RUN T - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 1 
                 NA 
                 20 
                 20 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 20 
                 20 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 20 
                 20 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 R01 
                 20 
                 20 
                 18 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 52.0 
               
               
                 1 
                 R02 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 77.0 
               
               
                 2 
                 R03 
                 20 
                 20 
                 22-20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 78.0 
               
               
                 2 
                 R04 
                 20 
                 20 
                 18 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 44.0 
               
               
                 2 
                 R05 
                 20 
                 20 
                 18 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 32.0 
               
               
                 2 
                 R06 
                 20 
                 20 
                 18 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 32.0 
               
               
                 2 
                 R07 
                 20 
                 20 
                 22-20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 30.0 
               
               
                 2 
                 R08 
                 20 
                 20 
                 18 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 26.0 
               
               
                 2 
                 R09 
                 20 
                 20 
                 18-20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 20.0 
               
               
                 2 
                 R10 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 22.0 
               
               
                 3 
                 R11 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 38.0 
               
               
                 3 
                 R12 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 34.0 
               
               
                 3 
                 R13 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 41.0 
               
               
                 3 
                 R14 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 21.0 
               
               
                 3 
                 R15 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 21.0 
               
               
                 3 
                 R16 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 21.0 
               
               
                 3 
                 R17 
                 20 
                 20 
                 20 
                 10/9.5 
                 0.9 
                 1.4 
                 1.0 
                 21.0 
               
               
                 3 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 Segment 1 Diagnostic 
               
               
                 Segment 2 Dryer Temperature &amp; Linespeed Variation 
               
               
                 Segment 3 Porosity and Thickness Variation - Starting Membrane 
               
             
          
         
       
     
     
       
         
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5D 
               
             
             
               
                   
               
               
                 MODIFICATION DATA FOR Hydrophilic PVDF Western Blotting Membrane 
               
             
          
           
               
                   
                   
                 Water 
                 Water 
                 Water 
                   
                 Opacity/ 
                 Salt 
                 Salt 
                 Salt 
                 Salt 
                   
                 QC 
                 Western 
               
               
                   
                   
                 Wet 
                 Wet 
                 Wet 
                 Speckle 
                 Trans- 
                 Wet 
                 Wet 
                 Wet 
                 Wet 
                 Fluores 
                 Blotting 
                 Blotting 
               
               
                 Run 
                 Roll 
                 Speed 
                 Uniform 
                 Through 
                 Level 
                 lucence 
                 Time 
                 Time 
                 Time 
                 Time 
                 BKG 
                 Pass/Fail 
                 Live 
               
               
                 Segment 
                 No. 
                 OK? 
                 X-web? 
                 OK? 
                 OK? 
                 OK? 
                 Seconds 
                 Seconds 
                 Seconds 
                 Seconds 
                 RFU 
                 Control 
                 Lysate 
               
               
                   
               
             
          
           
               
                 RUN T - POROSITY/THICKNESS, VMF4 DRYER TEMP, LINESPEED 
               
             
          
           
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 1 
                 R01 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 2.4/5.6  
                 3.4/6.2  
                 3.1/6.2 
                 3.0/6.0  
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 1 
                 R02 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 2.9/12.6 
                 3.2/11.4 
                 2.9/2.9 
                 3.0/9.0  
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R03 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 1.1/24.1 
                 1.4/10.8 
                 1.1/4.1 
                 1.2/13.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R04 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 1.2/14.5 
                 1.5/19.9 
                 1.3/5.6 
                 1.3/13.3 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R05 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 1.3/10.5 
                 1.5/15.4 
                 1.3/4.2 
                 1.4/10.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R06 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 1.3/24.7 
                 1.4/9.5  
                 1.3/4.0 
                 1.3/12.7 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R07 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 2.5/60.0 
                 2.3/25.4 
                 2.5/5.4 
                 2.4/30.0 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R08 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 2.1/9.7  
                 2.6/14.0 
                 2.6/5.5 
                 2.8/9.7  
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R09 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 2.8/13.1 
                 3.5/22.6 
                 2.5/5.9 
                 2.9/13.9 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 2 
                 R10 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 3.1/28.7 
                 3.5/15.7 
                 2.5/7.1 
                 3.0/17.2 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R11 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 24.1 
                 6.5 
                 4.2 
                 11.6 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R12 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 47.1 
                 35.7 
                 8.6 
                 30.5 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R13 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 67.9 
                 70.7 
                 34.5 
                 57.7 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R14 
                 Y 
                 Y 
                 Y 
                 N (HIGH) 
                 N (White) 
                 &gt;180 
                 &gt;180 
                 &gt;180 
                 &gt;180 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R15 
                 Y 
                 Y 
                 Y 
                 N (HIGH) 
                 N (White) 
                 &gt;180 
                 &gt;180 
                 &gt;180 
                 &gt;180 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R16 
                 Y 
                 Y 
                 Y 
                 N (HIGH) 
                 Y 
                 &gt;180 
                 57.3 
                 16.4 
                 &gt;180 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 R17 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
                 19.3 
                 40.8 
                 5.1 
                 21.7 
                 N/Avail. 
                 N/Avail. 
                 ≧NC 
               
               
                 3 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
               
                 Segment 1 Diagnostic 
               
               
                 Segment 2 Dryer Temperature &amp; Linespeed Variation 
               
               
                 Segment 3 Porosity and Thickness Variation - Starting Membrane 
               
             
          
         
       
     
     EXAMPLE 3 
     The protocol used for Western blotting and chemiluminescent detection is as follows:
         Protein samples are electrophoretically separated using Bis:Tris (4˜12%) midi gradient gel (Invitrogen, WG1402BOX).   Samples are electro-blotted at 45V for 1 hr 15 min using BioRad tank transfer apparatus (Criterion Blotter #165-6024) onto a hydrophilic membrane prepared as in Example 1.   Blots are washed 2× (3 min each) in TBS-T (0.1% Tween)   Blots are blocked for 1 hr at RT in TBS-T with 3% NFM (non-fat milk, Carnation).   Blots are washed 2× (3 min each) in TBS-T (0.1% Tween)   Blots are incubated with primary antibody in TBS-T for 1 hr.   Blots are washed 3× (5 min each) in TBS-T (0.1% Tween)   Blots are incubated with secondary antibody in TBS-T for 1 hr   Blots are washed 4× (5 min each) in TBS-T (0.1% Tween)   Protein bands are visualized using ECL (Millipore Immobilon-HRP) and x-ray film.       

     This protocol was used on the samples prepared in Example 2, and the blotting results are shown in  FIGS. 1 ,  2 , and  3 . 
     Western blotting results from a typical development run demonstrate the performance differences between the hydrophilic PVDF blotting membrane of this invention, and the controls (FL—hydrophobic PVDF membrane, and NC—Whatman/S&amp;S BA-85 membrane). Each horizontal strip in the figure contains 5 separate Western transfer blots; three blots on hydrophilic PVDF development samples, and one blot on each of the control membranes. Each strip of 5 Western blots is the result from one electrophoresis and transfer experiment (5 gels were run in each experiment). By design, each experiment embodies identical conditions with identical quantities of protein sample (applied in 4 lanes across each gel) before electrophoresis and transfer. The results shown are the transfer blots for the detection of 2 proteins (HSP70 and GAPDH) from a complex sample mixture (lysate), applied at decreasing sample solids (concentrations), from left-to-right and corresponding to: 5 ug, 2.5 ug, 1.25 ug, 0.67 ug. 
     Note that in each row (the result of a single electrophoresis and electro-blotting experiment, three hydrophilic blotting membranes of the invention are compared to two control blotting membranes. One control consists of nitrocellulose blotting membrane (NC) and the other control is a hydrophobic PVDF blotting membrane (FL). In the case of each single experiment, the FL membrane demonstrates the highest signal intensity for the 4 titers of analyte protein solution, and the NC membrane demonstrates the lowest signal intensity. It can be seen, when comparing the signal strengths between NC control blotting membranes and hydrophilic PVDF blotting membranes of the invention, that at the highest analyte sample titers (left hand side of each blot), that the NC and the hydrophilic PVDF membranes exhibit similar signal strengths. However, as one progresses to lower and lower analyte sample titers (progressing from left-to-right), the signal strength falls off more rapidly for the NC membrane. This demonstrates that the hydrophilic PVDF membrane allows sample detection at lower protein concentrations than NC membrane does.

Technology Classification (CPC): 6