Abstract:
A technique for producing multiple protein blots from a single gel, including entering the number ‘n’ membranes to be blotted into microprocessor memory, determining calibration constants for a gel batch, inputting calibration constants into microprocessor memory, and calculating ‘n’ voltage/time profiles for simultaneous blotting. A gel layer from the gel batch is treated with a protein sample, ‘n’ membranes are placed onto the gel layer to yield a gel pack, and the gel pack is placed between an electrode plate maintained at a fixed first voltage and an array of ‘n’ spaced generally parallel electrodes. The voltage to each respective ‘n’ spaced generally parallel electrodes is varied over time according to a respective ‘n’ voltage/time profile to transfer proteins to each respective ‘n’ membranes to yield blotted membranes to yield ‘n’ respective blotted membranes.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This patent application claims priority to co-pending U.S. Provisional Patent Application Ser. No. 61/407,253, filed on Oct. 27, 2010. 
    
    
     TECHNICAL FIELD OF THE INVENTION 
     The invention relates generally to the field of analytical biochemistry and, specifically, to a method and apparatus for simultaneously generating multiple protein blots. 
     BACKGROUND 
     The Western blot technique for detecting and identifying specific proteins in biological samples is commonly used among life scientists. This blotting technique utilizes gel electrophoresis to fractionate native or denatured proteins based on their migration speed (mobility) in a gel while under an electrical field. The proteins, trapped and size-fractionated in a gel, are transferred to and immobilized by a positively-charged membrane. (See  FIG. 1 ) Using primary antibodies specific to the target proteins, as well as secondary antibodies for signal amplification and visualization, expression and modification of the target proteins can be investigated. Although the procedure is well-established, and many tools are available for gel fractionation and blotting, one of the bottlenecks is its limited efficiency and controllability during the blotting procedure. 
     In most applications of Western blotting, there is a need to evaluate expression levels of multiple protein targets. Signal transduction pathways, for instance, are often activated by protein modifications such as glycosylation and phosphorylation. It is important to be able to determine the amount of signaling molecules that are, in many cases, phosphorylated. However, the current blotting technique can generate only a single membrane per gel, and thus life scientists usually have to prepare multiple gels. When the results from multiple gels are compared, one source of error may arise from potential chemical and physical variations among the gels. Furthermore, since the preparation of protein samples, running gels, and transferring to membranes are time consuming and costly, it is desirable to develop a blotting procedure that enables multiple blotting from a single gel. Such a procedure could reduce potential inconsistencies during sample loading, electrophoresis, and blotting, which are affected by varying factors including operational time, temperature, and chemical composition. 
     Thus, there is a need for a quicker and more efficient protein separation technique. The present novel technology addresses this need. 
     SUMMARY 
     The present novel technology relates to protein blotting. One object of the present invention is to provide an improved method and apparatus for generating protein blots. Related objects and advantages of the present invention will be apparent from the following description. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Schematic view of protein migrating through a gel during electrophoretic size fractionation (prior art). 
         FIG. 2 . Schematic view of a multiple anode array according to a first embodiment of the present novel technology. 
         FIG. 3A . Schematic view of a first embodiment blotting apparatus according to a first embodiment of the present novel technology incorporating the multiple anode array of  FIG. 2 . 
         FIG. 3B . Schematic view of an alternate embodiment blotting apparatus according to a second embodiment of the present novel technology incorporating a multiple cathode array. 
         FIG. 4A . Schematic view of the embodiment of  FIG. 3A  as connected to a microprocessor and power source. 
         FIG. 4B . Schematic view of the embodiment of  FIG. 3B  as connected to a microprocessor and power source. 
         FIG. 5 . Schematic view of  FIG. 2  superimposed with a diagrammatic view of protein separation. 
         FIG. 6A . Graph of signal intensities of β-actin with various blotting durations, representative blotting images after semi-dry transferring for 5, 13, 20, 40, 60, 90, 120 min. 
         FIG. 6B . Graph of normalized signal intensities as a function of 15 blotting durations, with maximum intensity set to 1. 
         FIG. 7A . Graph of multiple transfers using a non-uniform voltage profile, blotting images of three transfers, transfer time 14.5 min (1 st  transfer), 6.5 min (2 nd  transfer), and 15 min (3 rd  transfer). 
         FIG. 7B  Graph of differential transfer efficiency using a non-uniform voltage profile, white boxes indicate the expected positions of β-actin bands, in which the right box corresponds to ˜15 V, while the left ˜2 V does not show any visible band. 
         FIG. 8 . Schematic diagram of microcontroller circuits for generating a series of PWM signals for establishing a well-controlled voltage profile to an array of anodes, according to the embodiment of  FIG. 3A . 
         FIG. 9 . Schematic diagram of an amplifier circuit for multi-blotting according to the embodiment of  FIG. 3A . 
         FIG. 10 . Diagram of multi-blotting technique incorporating the system of  FIG. 2 . 
         FIG. 11 . Diagram of method of determining respective voltages to apply to respective voltages to apply to respective electrodes of system of  FIG. 2 . when applying technique of  FIG. 10 . 
     
    
    
     DETAILED DESCRIPTION 
     For the purposes of promoting an understanding of the principles of the novel technology and presenting its currently understood best mode of operation, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, with such alterations and further modifications in the illustrated device and such further applications of the principles of the novel technology as illustrated therein being contemplated as would normally occur to one skilled in the art to which the novel technology relates. 
     Blotting is a common protein transfer technique widely used for molecular analysis in the life sciences. Blotting involves the transfer of electrophoretically separated protein samples, immobilized in a polyacrylamide gel, to a blotting membrane. The present state of the art for the protein transfer is placing a gel, which has a mixture of proteins with various electrophoretic mobilities, in a constant electric field and generating one membrane per gel. When multiple blotting copies are needed as in many applications, multiple gels need to be prepared for each of the proteins to be analyzed. Two blotting copies from a single protein gel, for instance, can be used for identifying a total amount of proteins of interest as well as its specific subpopulation level, such as phosphorylated isoform. 
     As shown in  FIGS. 2-11 , the present novel technology relates to a technique  10  of producing multiple membranes  15  from a single gel, regardless of protein sizes. The first advantage of this multi-blotting technique  10  is that a user can produce multiple membranes  15  of identical protein samples. The ability of producing identical membranes  15  is useful, particularly in a comparative study, and also eliminates one source of error arising from chemical and physical differences. The second advantage of this method  10  is that it significantly reduces labor and chemical costs. 
     In order to uniformly transfer proteins regardless of their sizes, the novel technology includes 1 the introduction of a new multi anode array  20  (or multi cathode array  21 ) design for the application of varying voltages to different proteins during blotting, 2 a method  30  to determine different voltage levels to be applied to the multi anode array  20  (or multi cathode array  21 ), and 3 a method  40  to use pulse width modulated (PWM) DC voltages to provide different average voltage levels to the multi anode (or multi cathode) for blotting. 
     The multi anode array  20  enables the application of varying voltages to proteins of different sizes (i.e., mobilities). The mobility of any protein can be approximated to be proportional to the transfer voltage and exposure time. Introducing multiple anodes  45  allows the application of different respective voltages  50  and/or respective field exposure durations  55  for proteins of different mobility during blotting. 
     Typically, protein mobility is characterized by a previous step of protein fractionation  60  using electrophoresis in a vertical gel. In this vertical gel, proteins under the constant voltage move in the same direction with varying mobility (see  FIG. 2 ). Generally, smaller proteins have higher mobility in a given gel. 
     Description of Multi Anodes Plate 
     To generate multi blotting membranes  15  from a single gel  65 , an array  20  of multiple independent anodes  45  is positioned opposite a common cathode  20 , enabling the application of different voltage  50  and/or running time  55  for proteins of different sizes. The multi anode array  20  is arranged on a non-conductive plate  75 , as shown in  FIG. 2 . Each anode  45  is represented by a black stripe, and the insulated gap  80  between two adjacent anodes  45  by a white stripe. There is no theoretical limitation on the number of anodes  45  or the width of an anode  45 , but for generating a practical voltage profile the width of each anode  45  might typically be about 4 mm and the gap width 1 mm. Using a standard blotting protocol, a gel  65  and a blotting membrane  15  are configured as shown in  FIG. 3A  and  FIG. 3B . A higher voltage is typically applied to the anode  45  that is in electric communication with a group of larger proteins. 
     In order to apply well-controlled voltages to proteins in the gel  45 , protein mobility  85  is typically evaluated during size fractionation  60  using electrophoresis in a vertical gel  90 . This mobility  85  is affected by various factors  95  besides protein sizes  95 A, including gel composition  95 B and properties  95 C, buffer solution  95 D, electrophoretic voltage  95 E, running time  95 F, and the like. Electrophoretic voltage  95 E and running time  95 F are recorded and used for determining an appropriate voltage profile  100  for blotting. Likewise, gel and buffer solution factors  105 ,  95 B,  95 C,  95 D, are taken into account if they are going to differ between the fractionation pre-step system and the blotting system  110 . 
     One example theoretical basis for determining a non-uniform voltage profile for  106  multi-membrane blotting  10  for proteins with varying mobility follows. For a protein P migrating in vertical electrophoresis, when the time (t) is constant, the protein-moving distance (d) is proportional to the voltage; when the voltage is constant, the protein moving distance (d) is likewise proportional to the time (t). Therefore, the protein moving distance in vertical electrophoresis can be described by equation 1:
 
 d=kvt   (1)
 
where k is a coefficient constant, v is the voltage and t is time for blotting.
 
     The constant k is determined by three factors, the properties of the gel  65 , the protein sizes, and the electrical conductivity of the buffer solution  95 D. Equation (1) also may be applied in the horizontal blotting. In both vertical and horizontal systems  105 ,  110 , the buffer solution  95 D and the presence or absence of supporting matrices  115  and blotting membranes  15  may be different. Let the constant k for horizontal transfer be k h  and the constant k for vertical electrophoresis be k v , the ratio of a=k v /k h  is related to electrical conductivities of the electrophoresis system and is approximated to be a constant for all proteins. In common protein experiments, there are a small number of buffer solutions  95 D. Therefore, the constant a is readily determined. 
     Protein vertical migration may be modeled with d 1 =r*L at a given v 1  and t 1 , where d 1  is the migration distance and L is the maximum vertical travel length in the gel and r is a value between 0 and 100%. 
     If a protein moves a vertical distance d 1  in a blotting condition of v 1  and t 1  and the same protein moves a horizontal distance d 2  in a condition of v 2  and t 2 , according to equation 1, the following relationship is valid:
 
 d   1   :d   2 =( k   v   *v   1   *t   1 ):( k   h   *v   2   *t   2 )
 
           ( r*L ): d   2 =( k   1   *v   1   *t   1 ):( k   2   *v   2   *t   2 )
 
           k   v   *v   1   *t   1   *d   2   =k   h   *v   2   *t   2   *r*L  
 
           v   2   *t   2 =( k   v   *v   1   *t   1   *d   2 )( k   h   *r*L )
 
           v   2   *t   2 =( a*v   1   *t   1   *d   2 )/( r*L )  2
 
Note that v 1 , t 1  can be measured in vertical electrophoresis, L is the length of the gel, and d 2  is the thickness of the gel. Note also that in this case, k 1  and k 2  are equivalent to k v  and k h , respectively. The constant a can be predetermined experimentally. The equation 2 is valid for proteins of all sizes across the gel  65  and independent of gel types and protein types. There are two ways to use equation 2 to calculate the voltage and time applied to each anode in horizontal blotting.
       1. Regulate running time and constant voltage  55 .   2. Regulate transfer voltage with constant running time  50 .    By placing the maximum v 2  at a location r 1 *L, where r 1  is close to 0 (e.g., for r 1 =0.1), t 2  can be determined:
 
 t   2 =( a*v   1   *t   1   *d   2 )/( r*L*v   2 )=( v   1   *t   1   *d   2 )/ r*L*v   2Max )  3
 
After t 2  is determined, voltage v, at various points (r*L) for r from 0.1 to 1, can be determined:
       

     
       
         
           
             
               
                 
                   
                     
                       
                         
                           
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     Equations 2 and 4 provide the following information:
         a. v 2  decreases inversely proportional to r for 0.1&lt;r&lt;1.   b. Proteins of any size may move the same distance d 2  in time t 2 , and t 2  can be calculated based on equation 3.
 
For practical purpose v 2Max  can be assigned at the location of L*0.1(r 1 =r=0.1).
 
Generating Voltages and Time for Multi-Anodes Arrays
       

     Typically, it is undesirable to employ multiple DC voltage sources to generate the well-controlled voltage profile  100  during blotting, since a multiple source approach is not cost effective. Instead, it is more typical to use a single DC voltage source  120  operationally connected to a computer  125 , which generates multiple pulse width modulated (PWM) DC voltages  130  and produces the voltage signals  135  for each arrayed anode  45 . Each PWM voltage driving signal  135  is typically individually amplified, and all PWM signals  135  are typically amplified with the same gain. The duty cycle of each PWM  135  is determined to make the average PWM voltage  130  after amplification identical to the driving voltage  135 . The system block diagram is shown in  FIG. 5 . 
     If the amplifier gain is G, the peak voltage of all PWM after amplification is V, and the desired voltage  130  for the anode  45  is calculated based on equation 2 is v 1 . Then, the duty cycle of the voltage applied to anode I is:
 
PWM Duty cycle for Anode  I=[v   1 /( V/G )]*100%  5
 
The frequency of the PWM  130  may be in a wide range from KHz to a minimum 1/t Hz, where t is the time used in size fractionation using the vertical gel  90 . When the period of the PWM is set to 1/t Hz, the outcome is equivalent to the case in which a constant voltage is applied with varying time to individual anodes  45 .
 
Operational Procedure
 
     A typical experimental procedure (see  FIGS. 10 and 11 ) using the set-up, illustrated in  FIG. 3B , is:
         1. Size-fractionate proteins in electrophoresis with a vertical gel apparatus  105 .   2. Specify the vertical gel  90  running conditions (e.g., 150 V for 40 min with gel 1 mm thick, and constant a) as well a number of blotting membranes  15  to be made. The microcontroller  125  determines and generates the transfer conditions  100  (e.g., 3-30 V gradients for 10 min).   3. Place the gel  65  to a semi-dry transfer system.   4. Mount a blotting membrane  15  on the top of the gel  65 .   5. Conduct the transfer to the mounted membrane  15 ; transfer conditions  100  are automatically set in step 2.   6. After completing the transfer process  15  for the mounted membrane  15 , remove the membrane  15 ; mount a new membrane  15 , and conduct the transfer.   7. Repeat steps 4 to 6 for the number of blotting membranes  15  entered in step 2.
 
Variations
       

     The method described in section 2 can be achieved using multiple cathodes  46  with a common anode  71 . 
     The Description of Multi Cathodes Plate 
     In this method, multiple cathodes  46  are placed on a non-conductive plate  75  as shown in  FIG. 3A ,  3 B. There is no theoretical limit on the number of cathodes  46  and the width of each cathode  46 . For example, multiple voltages for multiple cathodes  46  can be arranged with the width of each cathode  46  as 4 mm and the gap  80  between two cathodes is 1 mm. 
     The placement of a gel  65  and a membrane  15  in blotting is shown in  FIG. 7A  and  FIG. 7B . The relative orientation of the gel  65  and the multiple anodes array  20  is shown in  FIG. 8 . Note that the blotting system  110  applies a higher voltage to the anodes  45  where large proteins are transferred. 
     Generating Voltages and Time for Multi Anodes 
     This invented method employs a single DC voltage source  120  and a computer  125  for generating multiple pulse width modulated DC voltages  130 , and thus a single source  120  produces all voltage signals  135  for all anodes  45 . Each PWM driven signal  135  is typically individually amplified, although all PWM signals  135  are amplified with the same gain. The duty cycle of each PWM is determined to make the average PWM voltage  130  substantially identical to the corresponding driving voltage. The system block diagram is shown in  FIG. 9 . 
     The amplifier gain is G, the peak voltage of all PWM after amplification is V, and the desired voltage for the anode I calculated based on equation 2 is v i , and the duty cycle of the voltage applied to cathode I becomes:
 
PWM Duty cycle for cathode  I==[V−v   i )/( V/G )]*100%  (6)
 
     Thus the instant technique is able to:
         1. Use multiple anodes  45  and a common cathode  70  (or the inverse  46 ,  71 ) for conducting semi-dry blotting and dry blotting.   2. Produce multiple membranes  15  using a single gel  65  that contains proteins of varying sizes.   3. Apply different voltages  100  on the multiple anodes  45  (or multiple cathodes  46 ) to ensure that proteins of different sizes can be uniformly transferred.   4. Generate different voltages  50 , which are applied to the anodes  45 , by multiple analog DC voltage sources or pulse width modulated DC voltages  130 .   5. Use different running times  55  for applying voltages on the multiple anodes  45  (or multiple cathodes  46 ) to ensure the proteins of different sizes can be transferred at the same efficiency.   6. Employ PWM to control the voltage  130  for blotting.       

     In one embodiment, a novel protein blotting device  110  is provided with two functions. First, the blotting system  110  is able to generate multiple (typically three) blotting membranes per gel  65  with equal quality (multi-blotting). Second, proteins with varying sizes (for instance, 20 to 150 kD) are transferred in the same percentage rate in the same blotting procedure without altering transferring time (uniform transferring). 
     The efficiency of protein transport was investigated by establishing a cumulative signal intensity function P(t), as a function of transferring time, t. For instance, P(t 1 )=⅓ indicates that one third of proteins transferred for a duration of t 1 . An electrophoretic voltage profile was derived along the directions of protein migration, in which one third of proteins regardless of their molecular size would be transferred in t 1 . This voltage profile was applied to a multi-anode plate using a micro-controller based pulse width modulated (PWM) voltage generator ( 120 ). 
     EXAMPLES 
     Western Blotting 
     Protein Samples 
     MC3T3 osteoblast-like cells were cultured in αMEM medium containing 10% fetal bovine serum and antibiotics (50 units/ml of penicillin and 50 μg/ml of streptomycin). Cells were incubated at 37° C. in a humid chamber with 5% CO 2  and prepared for experiments at 70-80% confluency. Protein samples were isolated by sonicating cells using a sonic dismembrator and lysed in a protein lysis buffer. 
     Electrophoresis and Semi-Drying Transferring 
     Isolated proteins were size-fractionated using 10% SDS gels (1.5 mm thickness). Electrophoresis was conducted using 100 V for 10 min (stacking), followed by 150 V for various durations (separation). Proteins, immobilized in SDS gels, were electro-transferred to Immobilon-P membranes using a semi-dry transferring apparatus. The standard transferring condition was 15 V for 40 min. To evaluate the effects of transferring conditions such as voltages and running times, varying transferring conditions were also examined. 
     Antibody Reactions and Image Analysis 
     The Immobolin-P membrane after protein blotting was incubated overnight in a blocking solution (1% milk in a PBS buffer). The membrane was then incubated for 1 hour with monoclonal β-actin antibodies followed by 45 min incubation with goat anti-mouse IgG conjugated horseradish peroxidase. The protein levels were assayed using an ECL Western blotting detection kit, and signal intensities were quantified using a luminescent image analyzer. 
     Protein Multi-Blotting Device 
     Prediction of the Blotting Voltage Profile 
     In order to uniformly transfer proteins regardless of their size in a given transferring time, blotting voltages were regulated for individual proteins. Based on observation in size fractionation, the mobility of proteins was assumed to be proportional to an applied electrophoretic voltage and transferring time. Through experimentation, the blotting voltage profile along the direction of protein migration was observed to achieve a uniform transfer of proteins any size. In the described blotting device, a protein with a normalized mobility of r would receive a local blotting voltage that was inversely proportional to r. 
     For a protein transferring in a gel, its moving distance, d, can be modeled to be proportional to the electrophorectic voltage, v, and blotting time, t:
 
 d=kvt   1
 
with k as a proportional factor. Given that for a fixed time, T, a moving distance is expressed as d=rL, where L=length of the gel, and r=normalized mobility ration between 0 (no mobility) and 1 (maximum mobility). In the described apparatus, any proteins have the same mobility along the thickness of the gel for a constant time with a graded electrophoretic voltage for individual proteins. Assume the largest protein of interest moves d min  at v max  with d min =r min L. Then, v for any protein with a normalized mobility ratio of r can be determined:
 
 v=v   max  
 
( r   min   /r )  2
 
     In summary, the graded electrophoretic voltage in the described apparatus is regulated to be inversely proportional to its normalized mobility rate. 
     Design of the Multiple Anodes and the Microcontroller Base PWM Generator 
     In one embodiment, the multi-blotting device  110  has a plate with multiple anodes  45  ( FIG. 1 ). With proper regulation of blotting voltage profiles, proteins of the identical molecular size receive the same voltage, while larger proteins are transferred with the higher voltage. Here, a single DC voltage source  120  and a computer-controlled  125  voltage generator provided voltages for all anodes  45  ( FIG. 2 ). This generator provides multiple pulse width modulated (PWM) DC signals  135  of different duty cycles for all anodes  45 , in which each PWM signal  135  was individually amplified by a PWM amplifier  140  and applied to one anode  45 . The voltage amplification gains are the same to all PWM signals  135 . The duty cycle was set to make the average voltage of an amplified PWM equal to the desired driving voltage of the corresponding anode  45 . Assuming the amplification gain is G, the peak voltage of all PWM after amplification is V, and the desired voltage for anode I is v i , the duty cycle of the voltage applied to anode I is [v i /(V/G)]*100%. 
     Examples 
     Experimental Evaluation and Validation 
     Blotting Time and Signal Intensities 
     Prior to generating multiple blotting membranes  15  from a single gel  65 , a relationship of signal intensities to transferring time was built by conducted semi-dry blotting using various transferring times  55 . Taking β-actin as an example, representative blotting images  145  are shown for blotting for 5, 13, 20, 40, 60, 90, and 120 min at 15 V ( FIG. 6A ). The two protein bands in each image  145  were generated using the identical conditions. The signal level after reactions with antibodies was initially increased as the blotting time was lengthened. However, the level was saturated for the transfers for 40 min or longer durations  55 . The normalized signal intensities showed that this temporal signal profile was approximated by a cumulative Gaussian distribution function (mean=22 min, and standard deviation=9 min) ( FIG. 6B ). This cumulative distribution function provided the basis for determining transferring time for each of the multiple blotting membranes  15 . Typically, if this cumulative function is a linear function of transferring the same, the same blotting time is applied to all membranes. The Gaussian distribution implies that for generating 3 membranes, the 1 st  and the 3 rd  transferring durations  55  are identical. The 2 nd  transferring duration  55  is calculated 83% of the standard deviation (approximately 7.5 min in our experimental setup). 
     Multiple Transfers 
     Based on the relationship between blotting time  55  and signal intensity  50  in the previous experiment, multiple transfers were next conducted (generation of 3 blotting membranes  15 ) using a single gel  65 . Selecting β-actin, the transfer time was chosen to be 15 min (1 st  transfer), 7 min (2 nd  transfer), and 15 min (3 rd  transfer). The result clearly shows that by properly choosing blotting durations  55  it is possible to generate multiple blotting membranes  15 , although there are variations in signal intensity among three membranes  15  ( FIG. 7A ). In each image, two protein bands were processed using the same experimental condition. 
     Transfer Under a Non-Uniform Voltage Profile 
     The experiments above support the first design criterion of achieving multiple blotting membranes  15  from a single gel  65 . To examine the second design criterion of simultaneously transferring proteins with various sizes, 40 min semi-dry blotting was conducted using a non-uniform voltage profile  100  ( FIG. 7B ). In this experiment all conditions were identical to the condition employed for generating the results depicted in  FIG. 3  except the voltages  50 . The voltages  50  applied to four different bands (left to right) were 12.0 V, 3.9 V, 2.3 V, and 1.7 V, respectively. The result using β-actin as an example demonstrates that transfer efficiency is controllable by an applied electrophoretic voltage  100 . The left position under 12 V exhibits a clear protein band, but the right position under approximately 1.7 V barely shows a detectable protein band. 
     Protein Multi-Blotting Device 
     Design and Prototyping the Multi-Anode Plate 
     An apparatus  100  was made including microcontroller  125   m  connected to multiple anode plate  20 , where the width of each anode stripe  45  was set to 4 mm and the width of the gap  80  between two respective anodes  45  was 1 mm. The size of both anode  20  and cathode plates  70  were 18 cm×24 cm, and the device  110  was designed to accommodate gels  65  with size up to 14 cm×17 cm. 
     Design and Prototyping Multi-Blotting Control Circuits 
     A PICF184515 microcontroller-based voltage generator  120  was designed and prototyped to provide the designated voltage profile  100  to individual anodes  45  (see  FIG. 8 ). The microcontroller generated PWM voltage signals  135  were amplified. The average voltage of the amplified PWM signal  135  corresponded to the desired voltage for the anode  45 . 
     The maximum voltage of all PWM signals  135 , generated by the microcontroller  125 , was 5 V with the current of 100 μA. Since the PWM signals  130  did not provide the necessary power for driving a protein transfer, these signals were amplified using a TC 4469 MOSFETS amplifier  140  (see  FIG. 9 ). The maximum voltages of all PWM signals  130  were set to 15 V. 
     During blotting, as illustrated in  FIGS. 10 and 11 , the microcontroller device  125  was configured to request a user to provide the conditions used for protein fractionation with the vertical gel  90  as well as the conditions  95  for protein blotting. The former fractionation conditions  95  included length and the thickness of the gel  95 C, electrophoretic voltage  95 E, and running time  95 F. The latter transferring conditions  95 E were the power supply voltage, the number of membranes to be blotted, and buffer solution type  95 D used in horizontal blotting and vertical gel  90 . The program in the microcontroller  125  calculated the voltage levels needed for each anode  45  and the time required  55  for transferring proteins. Based on the mean and the s.d. values of Gaussian distributions, the blotting time  55  required for each membrane  15  was determined. The program gave a sequence of instructions and guide to a user such as “place the membrane on the device,” “start blotting,” “wait,” “change the membrane,” and the like. 
     The above-described a novel protein multi-blotting device  110  incorporates the multi-anode array  20  and the microcontroller-based voltage-regulating circuits  125 . In experimental evaluations, blotting signal intensities were modeled as a cumulative Gaussian distribution function of transferring durations and demonstrated that it was possible to generate 3 blotting membranes  15  from a single gel  65 . In the device development phase, an appropriate electrophoretic voltage profile  100  was derived and implemented using the multi-anode plate  20  and the microcontroller based PWM voltage generator  120 . The results, using β-actin as an example, supported the notion that the described device  110  was capable of providing superior quality for comparing the level of various proteins, reducing the required amount of samples, time, and cost for protein blotting. In generating a transferring voltage profile  100 , it was assumed that protein mobility was proportional to applied voltage and transferring time  55 . In order to regulate differential mobility of proteins with varying sizes, the new multi-blotting device  110  generated a well controlled voltage profile  100  in which individual proteins received transferring voltage inversely proportional to its intrinsic mobility. Although this particular design incorporates multi-anodes  20 , it is equally possible to build a device using multi-cathodes  21  and a common anode  71 . 
     In predicting transferring time for each of the multiple membranes  15 , a protein mobility model using a Gaussian function was constructed. Although average mobility can be characterized by blotting voltage  50  and running time  55 , any proteins of interest with same size present a variation in their mobility. Based on experimental results, this variation was approximated by a Gaussian distribution and the amount of proteins transferred to a membrane was predicted from a cumulative Gaussian distribution. By determining the mean and s.d. values, it was possible to divide the transfer process into multiple segments, each of which would receive the equal amount of proteins of interest. 
     In one embodiment apparatus  110 , copper was used as material for the anode  45  and stainless steel for the cathode  70 . It was observed that electrical plating occurred and a noticeable amount of cooper was transferred along with proteins to the cathode  70 . Other materials such as stainless steel or platinum-titanium alloy may be used to avoid the observed electrical platting during the blotting process. 
     In predicting and evaluating the capability of the device, commonly used materials and conditions such as 10% SDS gel, a transfer buffer with 70% methanol, and the like were selected. With fine-tuning, the voltage profile  100  and running time  55  for other materials and conditions may be determined. Although the focus of the operation of the described device was on semi-dry blotting, the same principle should apply to dry blotting. 
     While the novel technology has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character. It is understood that the embodiments have been shown and described in the foregoing specification in satisfaction of the best mode and enablement requirements. It is understood that one of ordinary skill in the art could readily make a nigh-infinite number of insubstantial changes and modifications to the above-described embodiments and that it would be impractical to attempt to describe all such embodiment variations in the present specification. Accordingly, it is understood that all changes and modifications that come within the spirit of the novel technology are desired to be protected.