bluuebunny/crossref_metadata_2025 huggingface dataset:
A PLS kernel algorithm for data sets with many variables and fewer objects. Part 1: Theory and algorithm
Authors: Rännar Stefan , Lindgren Fredrik , Geladi Paul , Wold Svante
Published: May 2005

A fast PLS regression algorithm dealing with large data matrices with many variables (K) and fewer objects (N) is presented For such data matrices the classical algorithm is computer‐intensive and memory‐demanding. Recently, Lindgren et al. (J. Chemometrics, 7, 45–49 (1993)) developed a quick and efficient kernel algorithm for the case with many objects and few variables. The present paper is focused on the opposite case, i.e. many variables and fewer objects. A kernel algorithm is presented based on eigenvectors to the ‘kernel’ matrix XX TYYT, which is a square, non‐symmetric matrix of size N × N, where N is the number of objects. Using the kernel matrix and the association matrices XXT (N × N) and YYT (N × N), it is possible to calculate all score and loading vectors and hence conduct a complete PLS regression including diagnostics such as R2. This is done without returning to the original data matrices X and Y. The algorithm is presented in equation form, with proofs of some new properties and as MATLAB code.
Generalized rank annihilation method. I: Derivation of eigenvalue problems
Authors: Faber N. M. , Buydens L. M. C. , Kateman G.
Published: May 2005

Rank annihilation factor analysis (RAFA) is a method for multicomponent calibration using two data matrices simultaneously, one for the unknown and one for the calibration sample. In its most general form, the generalized rank annihilation method (GRAM), an eigenvalue problem has to be solved. In this first paper different formulations of GRAM are compared and a slightly different eigenvalue problem will be derived. The eigenvectors of this specific eigenvalue problem constitute the transformation matrix that rotates the abstract factors from principal component analysis (PCA) into their physical counterparts. This reformulation of GRAM facilitates a comparison with other PCA‐based methods for curve resolution and calibration. Furthermore, we will discuss two characteristics common to all formulations of GRAM, i.e. the distinct possibility of a complex and degenerate solution. It will be shown that a complex solution‐contrary to degeneracy‐should not arise for components present in both samples for model data.
Electrochemical detection as an alternative to UV in RP‐HPLC peptide mapping
Authors: Chen L. , Krull I. S.
Published: May 2005

This article describes the possibility of electrochemical (EC) detection as an alternative to ultraviolet (UV) detection doing peptide mapping. The high sensitivity and selectivity of the electrochemical detection can be applied to determine some digested peptides. This approach demonstrated that peptides containing electroactive amino acids can be determined by EC detection. Furthermore, upon irradiation, nonelectroactive amino acids and peptides can be made electroactive, thereby increasing the sensitivity and selectivity of peptide mapping. A variety of effects are discussed to help in understanding the possibility of this useful approach.
Complexation of trialkyllead with diethyldithiocarbamate
Authors: Mikac Nevenka , Branica Marko
Published: May 2005

Sodium salt of diethyldithiocarbamate (NaDDC) is one of the best‐known organic reagents which form extractable chelates with many metal ions. Although many data on the characteristics of metal‐DDC complexes have been published, no information about organometal complexes is available. In this work, the calculation of stability constants of trialkyllead‐DDC complexes is described and their values are measured by electrochemical techniques. Obtained values are (1.9 ± 0.7) × 106 for (C2H5)3PbDDC and (3.1 ± 1.5) × 105 for (CH3)3PbDDC. The application of these constants for the prediction of trialkylleads behavior during complexometric extraction from water to organic solvent is discussed.
Electrode modification with a poly(NiII‐tetramethyldibenzotetraaza[14]annulene) film. Electrochemical behavior and redox catalysis in alkaline solutions. I
Authors: Cataldi Tommaso R. I. , Centonze Diego , Ricciardi Giampaolo
Published: May 2005

The voltammetric behavior in alkaline solution of a nickel‐based chemically modified electrode (Ni‐CME) prepared by oxidative electropolymerization of a nickel–tetraazaannulene complex (NiIIL) was investigated. After electrochemical deposition of the (NiIIL)n film in dichloromethane on a conducting substrate, the modified electrode was continuously cycled in alkaline solution until a steady voltammetric profile was obtained. Although upon electrochemical treatment in basic solutions the nickel–nitrogen tetracoordination of the (NiIIL)n film is probably lost, the nickel species remain entrapped inside the polymeric skeleton and exhibit stable redox behavior resembling that of the nickel hydroxide electrode. This makes it possible to prepare very thin films of nickel‐based modified electrodes. In cyclic voltammetry experiments the Ni‐CME exhibits a pH dependence of the peak potentials typical of the α‐Ni(OH)2/γ‐NiOOH redox process, and a separation between anodic and cathodic peak potentials of 64mV at pH 13.2 and 10 mVs−1. The redox catalysis at Ni‐CMEs of representative compounds that are not easily electrooxidized, such as choline, cadaverine, γ‐amino butyric acid (GABA), benzyl alcohol, and glycine, is demonstrated. The good stability of these nickel‐based modified electrodes makes them attractive in analytical applications.
Development of analytical procedures based on abrasive stripping coulometry and voltammetry for solid state phase microanalysis of natural and synthetic tin‐, arsenic‐, and antimony‐bearing sulfosalts and sulfides of thallium, tin, lead, and silver
Authors: Zhang Song , Meyer Birgit , Moh Günter H. , Scholz Fritz
Published: May 2005

Analytical procedures are described for the solid state phase microanalysis of synthetic and natural tin‐, arsenic‐, and antimony‐bearing sulfosalts and sulfides of thallium, tin, lead, and silver. In these procedures, abrasive stripping voltammetry and coulometry of the solid phases are applied. Trace amounts of the sample compounds were transferred onto the surface of a graphite electrode and the electrochemical reduction or oxidation was followed by voltammetric techniques. The most valuable technique for distinguishing different mineral phases is to record the anodic dissolution voltammograms after a preliminary electrolytic reduction of the sulfides or sulfosalts to the elementary metals which are still confined to the electrode surface. For various compounds an approach was developed in which the ratio of charges consumed during reduction and oxidation of the solid phases is used for unambiguous identification. Coulometric measurements are also used for the determination of the quantitative composition of the solid phases.
Determination of penicillin by triangle programmed coulometric acid–base flow titration in a system incorporating an enzyme reactor
Authors: Fehér Zsófia , Nguyen T. Len , Nagy Géza
Published: May 2005

A flow‐through coulometric titration technique is described which can be applied for the determination of a wide variety of compounds. If necessary, the sample can be transformed in the measuring system into a form which can be titrated. Thus an enzyme‐catalyzed reaction can also be used as a sample pretreatment step. In this article a measurement technique and apparatus are described in which an enzyme‐catalyzed reaction is used for the fast and reliable determination of penicillin compounds. The principle of the technique is that an enzyme reactor is built into the sample line of the measurement system used for performing coulometric triangle programmed titrations. One of the components of the reaction mixture leaving the reactor (in this case penicilloic acid) is determined by coulometric acid–base titration. This technique is especially useful for the analysis of large sample series.
Amplified biosensing of alcohol based on biocatalytic accumulation of the mediator
Authors: Wang Joseph , Naser Najih
Published: May 2005

A novel strategy for enhancing the sensitivity of alcohol‐dehydrogenase amperometric biosensors, based on biocatalytic accumulation of the mediator, is described. By performing the biochemical/chemical step under open‐circuit conditions, it is possible to accumulate the reduced form of the Meldola blue mediator, prior to the chronoamperometric quantitation of the enriched layer. Such temporal separation of the recognition and transduction events offers a 35‐fold sensitivity enhancement (following a 5‐min accumulation). Factors influencing the response are explored and applicability to various wine samples is illustrated. The new amplification strategy can be extended to biosensors based on other nicotinamide‐cofactor dependent dehydrogenases.
Electrochemical behavior of aromatic amines protected by the nitrobenzenesulfonyl group
Authors: Zanoni Maria V. Boldrin , Stradiotto Nelson Ramos
Published: May 2005

The electrochemical reduction of aniline protected by the 4‐, 3‐, or 2‐nitrobenzenesulfonyl group in N,N‐dimethylformamide (DMF) at a vitreous carbon electrode is reported. The compounds are reduced in three cathodic steps. The first reduction step at about −0.85 V vs. SCE occurs with the formation of an unstable anion radical, which decomposes via NH bond cleavage. Reduction of this sulfonamide anion occurs at −1.16 V for para and meta isomers and at −1.48 V for the ortho isomer. The third cathodic step occurs at higher cathodic potentials. The BH bond cleavage is always the preponderant process. The mechanisms of the reduction are discussed.
Effect of hydrogen fluoride on the anodic behavior of glassy carbon in aqueous and mixed aqueous solutions
Authors: Noel Michael , Santhanam Raman , Ravikumar Musuwathi K. , Flora Mangalam Francisca
Published: May 2005

The electrochemical behavior of the glassy carbon electrode (GCE) in aqueous 1.0–20.0 M HF solutions and in mixed solvents containing 15.0 M HF (25 vol.%) was investigated using cyclic voltammetry, chronoamperometry and surface microscopic analysis. The GCE shows three surface redox peaks in aqueous HF solutions which become distinct at higher HF concentrations. Fresh surface oxidation and slight surface roughening occurs above 1.6 V. The background current increases significantly due to strong anodic polarisation in HF solutions. However, the electrode activity as measured by the response of the ferricyanide/ferrocyanide redox system remains unaltered. In mixed solvent systems, the surface redox currents and fresh surface oxidation decrease significantly. The oxygen evolution occurs at less positive potentials. Thus the electrochemical activity in such systems is much better.
Surface charge density measurements with a controlled growth mercury electrode
Authors: O'Dea John J. , Ciszkowska Malgorzata , Osteryoung Robert A.
Published: May 2005

The surface charge density of the mercury‐electrolyte interface is estimated using chronocoulometry at a controlled growth mercury electrode. After initial formation and equilibration the mercury drop is expanded by further addition of mercury. Direct measurement of the charge required as new area is formed is used to estimate the surface charge density. The mercury drop is modeled as a step‐wise expanding sphere.

The capillary noise continuously produced by stationary drops under potential control was investigated and characterized. Spectral analysis of the noise reveals that the electrode is particularly sensitive to vibrations near the resonant frequency of the suspended drop. Ambient vibrations in the laboratory environment produce alternating currents at the electrode which vanish at the point of zero charge and so mark its position.
Multichannel electrochemical analyses of enzyme reactions
Authors: Kasai Nahoko , Matsue Tomokazu , Uchida Isamu
Published: May 2005

Horseradish peroxidase (HRP) and xanthine oxidase (XOD) reactions were investigated using a multichannel electrochemical detector with 16 independent band electrodes held at different potentials. Although the enzyme reaction media gave complicated electrochemical responses in flow injection analysis, responses for a redox species can be extracted easily from the multichannel responses. The course analysis over time of the extracted responses enabled the Michaelis constants and molecular activities of HRP for various electron donors to be established. The electrochemical responses of O on the μM order in aqueous solution were also washed out from the responses in the XOD reaction media.
Electroreductive cleavage of propargylic acetates through polarity inversion of in situ generated allenyl palladium(II) complexes and, in part, unusual chemical reduction induced by phosphine ligand
Authors: Tanaka Hideo , Takeuchi Hiroaki , Ren Qinghua , Torii Sigeru
Published: May 2005

The electroreduction of propargyl acetates in the presence of a catalytic amount of PdCl2(PPh3)2/PPh3 in DMF containing Et4NOTs at ambient temperature under a constant current density (10 mA/cm2) afforded the corresponding allenes in 43–84% yields. The time‐course of the reaction was monitored by HPLC, indicating that the allenes were formed immediately after the current was applied and that most of the starting materials were consumed when less than the theoretical amount of electricity (2 F/mol), typically 1.2–1.7 F/mol, was reached. Interestingly a considerable amount of phosphine oxide was formed in the electroreduction media. Neither the reductive cleavage of the propargylic acetates nor the formation of phosphine oxide occurred without electricity being applied. The unusual results suggest that chemical reduction with the phosphine ligand would take place, in part, during the course of the electroreduction. A plausible reaction mechanism is proposed involving electroreductive polarity inversion of in situ generated allenyl PdII‐complex as well as chemical reduction with the triphenylphosphine ligand leading to allenyl anion species.
Application of poly(cyclophosphazene) for potentiometric detection of tributyl phosphate vapor
Authors: Li Jing , Janata Jiří , Josowicz Miru
Published: May 2005

Potentiometric sensing layer for detection of tributyl phosphate (TBP) in the concentration range from 1.1 ppb up to 8.8 ppm is described. The high sensitivity to TBP is based on a specific charge‐transfer interaction that occurs between iodine species and the oxygen atom of the phosphoryl group of TBP. The iodine is incorporated electrochemically or chemically into a chemically inert poly(cyclophosphazene‐benzoquinone), PPBQ. matrix. The bulk involvement of the new material in the interaction with the TBP vapor leads to modulation of its Fermi level. The number of' moles of charge shared by the TBP with thc sensitive layer is between 0.8 to 0.9 in the range of concentrations between 0.27 and 8.8 ppm. The interaction is highly reversible and reproducible.
Ultrasonic effects on electroorganic processes. Part 4: Theoretical and experimental studies on product‐selectivity in electroreduction of benzaldehyde and benzoic acid
Authors: Atobe Mahito , Matsuda Kazuya , Nonaka Tsutomu
Published: May 2005

Product‐selectivity in the electroreduction of p‐methylbenzaldehyde and benzoic acid under ultrasonic irradiation are examined theoretically and experimentally on the basis of mass transport phenomena in lhe electrode interface. As a result, it was suggested that the selectivity is controlled by mass transfer coefficients which are greatly affected by cavitation and/or agitation effects of ultrasounds.
Determination of tin(II) in pharmaceuticals by amperometric oxidation after complexation with tropolone
Authors: Boutakhrit K. , Quarin G. , Özkan S. A. , Kauffmann J.‐M.
Published: May 2005

Inorganic tin(II) was determined, rapidly and selectively in the presence of tin(IV), by electrooxidation after complexation with tropolone (2‐hydroxy‐2,4,6‐cycloheptatrienone). By cyclic voltammetry, the tin(II)‐tropolone complex showed a well‐defined irreversible oxidation peak which occurred at −0.014 V (vs. Ag/AgCI) at the glassy carbon electrode in 0.05 molL−1 acetate buffer pH 5.5. The determination of SnII has been realized using FIA and by poising the electrode at +0.1 V. A linear relationship was obtained between peak current and tin(II) in the concentration range 0.25–20 μmolL−1. The detection limit was 0.1 μmolL−1 and the RSD calculated by injection of a 5 μmolL−1 tin(II) solution was 1.5% (n = 10).The optimization of several experimental parameters bas been realized and the influence of many cations and possible interfering species encountered in radiopharmaceuticals and in dental gels has been investigated. The method was applied to the determination of tin(II) in dental gels.
Amperometric alcohol sensors based on protein multilayers composed of avidin and biotin‐labeled alcohol oxidase
Authors: Du Xiao‐yan , Anzai Jun‐ichi , Osa Tetsuo , Motohashi Ryoichi
Published: May 2005

Protein multilayers composed of avidin and biotin‐labeled alcohol oxidase (AOx) were prepared on the surface of a platinum (Pt) electrode by repeated deposition of the proteins to develop amperometric alcohol sensors. The AOx multilayer‐modified sensors showed a rapid response to ethanol over the concentration range of 0.01–10mg/100mL. The output current of the sensor depended linearly upon the thickness of the AOx layers (or the number of the AOx layers), suggesting that a constant amount of AOx can be immobilized by each deposition. The selectivity of the sensor to aliphatic alcohols was in the order of methanol > ethanol > n‐propanol > n‐butanol, being in accord with the selectivity of AOx in a homogeneous system itself. The sensor retained its original response to ethanol for about one month without deterioration.
The transfer of chloride ion across an anion exchange membrane
Authors: Wang Hanming , Yu Zemu , Wang Erkang
Published: May 2005

The transfer of chloride ions into a low resistance anion exchange membrane (AEM) was investigated by cyclic voltammetry (CV) and electrochemical impedance spectra. In all cases, concentration polarization of C1− ions is exterior to the membrane. It controls the flux and produces the limiting currents: either steady state or transient (peak type) current. In CV experiments, when the size of the holes in the membrane was much smaller than the distance between membrane holes, the C1− anion transfer showed steady state voltammetric behavior. Each hole in the membrane can be regarded as a microelectrode and the membrane was equivalent to a microelectrode array in this condition. When the hole in the membrane was large or the distance between membrane holes was small, the CV curve of the C1− anion transfer across the membrane showed a peak shape, which was attributed to linear diffusion. In AC impedance measurement, the impedance spectrum of the membrane system was composed of two semicircles at low DC bias, corresponding to the bulk characteristics of the membrane and the kinetic process of ion transfer, respectively. The bulk membrane resistance increases with increasing DC bias and only one semicircle was observed at higher DC bias. The parameters related to kinetic and membrane properties were discussed.
Electrofocusing on flat pH gradients: Systematic pH gradient modification leading to improved protein separation
Authors: Nguyen Nga Y. , Chrambach Andreas
Published: May 2005

A pH gradient providing maximal separation between pH 4.7 and 4.9 was generated by systematic variation of carrier constituent mixtures, and/or selection of anolyte/catholyte. In the optimized pH gradient, separation on gel electrofocusing between proteins with pI's between 4.7 and 4.9 was shown to be superior to that achieved on relatively steep pH gradients. This was the case in the separation of either human growth hormone isohormones D and E, or of bovine serum albumin and ovalbumin.
Ultrathin‐layer isoelectric focusing in 50–100 μm polyacrylamide gels on silanized glass plates or polyester films
Authors: Radola Bertold J.
Published: May 2005

Ultrathin‐layer isoelectric focusing in 50–100 μm polyacrylamide gels covalently bound to glass plates or polyester films, pretreated with methacryloxypropyl‐trimethoxy‐silane, is described. The gels adhere firmly to the silanized supports during all steps of operation and visualization. Ultrathin gels are prepared by the new and simple “flap technique”. Artificial mixtures of marker proteins and crude fungal enzymes were focused on 5–25 cm separation distances. In 11 cm gels containing pH 4–9 “Servalyt T” carrier ampholytes, 2–3 zones per mm can be resolved, with a zone width of 70–150 μm and differences in pI values of 0.023–0.033 pH. The gel volume per focused sample is 15–120 μl and up to 100 samples can be analyzed on 12.5 × 26 cm gels. Polyacrylamide gels of intermediate and high porosity, e. g. 5 % T, 3 % C and 3 % T, 20 % C, are compared. Small marker proteins attain equilibrium in both gels after focusing for 1300–2000 V x h at a final field strength of 100 V/cm, whereas thyroglobulin (690 000) and ferritin (465 000), applied at the anode and cathode, coalesce only in high‐porosity gels. pH Gradients were determined, with the aid of a glass electrode, directly on the gel surface for wide range carrier ampholytes “Servalyt T”, “Ampholine”, and “Pharmalyte” after focusing for 2000–5000 V × h. Identical focusing patterns are found for some marker proteins and crude fungal enzymes in different carrier ampholytes. The effect of purification of commercial carrier ampholytes by ultrafiltration and charcoal treatment, and of “forced aging” at 80°C, on isoelectric focusing of marker proteins was studied. The molecular size distribution of different carrier ampholytes was assessed by thin‐layer gel chromatography on Bio‐Gel P‐10. Protein staining with Serva Blue G, Serva Blue R and Serva Violet 49 is described. The sensitivity of protein detection is 50–100 ng/mm2. Fixation, staining and destaining can be completed within 10–15 min in 50 μm gels. Ultrathin‐layer isoelectric focusing on silanized glass plates or polyester films recommends itself as an excellent analytical technique for research and routine applications. The method combines high resolution, speed, versatility and reagent economy with simplicity of operation.
Flattening, shifting and comparing pH gradients in slab gel electrofocusing by local increases of gel volume
Authors: Altland Klaus , Kaempfer Michael
Published: May 2005

A simple and versatile technique of horizontal slab gel electrofocusing is described which is based on a discontinuity of the gel volume along the pH axis achieved by overlaying ampholyte containing gel strips, of a defined volume and shape, at various locations on a base gel during the prerun of isoelectric focusing. In the electric field the overlaid gel strip delivers ampholytes of a distinct pI interval into the strip‐free part of the base gel and removes from the latter some ampholytes present in the system. Flattening of the pH gradient in the strip‐free part of the gel is the consequence. The technique is especially useful for thin gels and can be performed with any commercially available horizontal slab apparatus.
A comparison of crossed immunoelectrophoresis and crossed immunoelectrofocusing for the investigation of human salivary proteins
Authors: Eckersall Peter D. , Beeley Josie A.
Published: May 2005

Using two dimensional immunological electrophoretic techniques, it is possible to study the antigens in microlitre quantities of saliva without prior concentration. Under the conditions used, crossed immunoelectrophoresis was superior to crossed immunoelectrofocusing in that the former revealed a greater heterogeneity of salivary components. Both methods showed that there is considerable variation between individuals in the proportions of the different antigens present. Levels of some of the salivary antigens in a single individual were consistent between samples collected at different times of day. α‐A mylase was the major antigen of parotid saliva while the submandibular/sublingual secretion contained a wider spectrum including a major acidic antigen.
Principles and instrumentation of analytical cell electrophoresis
Authors: Catsimpoolas Nicholas
Published: May 2005

Measurement of the electrophoretic mobility of cells can be performed with various types of apparatus. Basically, these instruments can be classified into two broad categories i.e. those where physical separation does not take place and those which actually separate cell populations according to their surface charge. The former category includes the microscopic/image analysis method and the laser light scattering Doppler velocimetry apparatus. These two types of instruments can be used only for analytical purposes. Physical separation combined with analytical capabilities requires some form of anti‐convection stabilization of the separated species. This can be accomplished by capillary action, mechanical or electromagnetic rotation, and flowing or stationary density gradients. Several instruments exist which utilize each of the above principles. Monitoring of the separation is usually performed by optical scanning.
Purification of pepsinogen I from human urine by means of DEAE‐chromatography and isotachophoresis
Authors: Axelsson Christen Kirk , Axelsen Nils Holger , Svendsen Per Just
Published: May 2005

The present paper gives a description of a procedure for isolation of pepsinogen I from human urine. The initial steps are concentration by batch adsorption to DEAE‐Sephadex, desorption by addition of NaCl, and DEAE‐chromatography. The final step is preparative isotachophoresis with Ampholine carrier ampholytes as spacers. Exact visualization of the fractionations is obtained by fused rocket immunoelectrophoresis. The procedure provides pepsinogen I of high purity as shown by crossed immunoelectrophoresis, agar gel electrophoresis and N‐terminal aminoacid analysis. The yield of the procedure is 0.05–0.1 mg pepsinogen I per liter urine.
Assay of UDP‐glucuronyltransferease with mixtures of acceptors using analytical capillary isotachophoresis
Authors: Holloway Christopher J. , Husmann‐Holloway Sabine , Brunner Gorig
Published: May 2005

Analytical capillary isotachophoresis has been applied as an analytical tool for the assay of the microsomal enzyme UDP‐glucuronyltransferase, which is an important system for the detoxification of drugs and many endogenous and exogenous toxins. The substrate donor molecule UDP‐glucuronic acid, various products of hydrolytic cleavage, and the products of glucuronidation β‐glucuronides, can be assayed in a single run. Furthermore, the glucuronidation of mixtures of acceptors can be monitored simultaneously, since the various β‐glucuronides are separated from one another. This method offers a more comprehensive assay of glucuronidation reactions than has been possible using previously published procedures.
Gel electrophoresis salivary mucins
Authors: Kashket Shelby , Ciociolo James M.
Published: May 2005

Bovine Submaxillary mucin (BSM) and human submandibular mucin (HSM) were electrophoresed in a system in which the mucins were incorporated into an agarose sample gel and separation was carried out in a 5 % polyacrylamide gel. BSM was resolved into two protein‐ and carbohydrate‐containing bands, and a number of non‐mucin proteins. When the electrophoresis was carried out at 80°C, the mobilities and resolution of the bands were increased. The entry of HSM into the running gel was also increased at the elevated temperature and the samples were separated into at least four protein‐ and carbohydrate‐stainable bands. Protein bound sialic acid remained unchanged unter these conditions and aggregating activity for cells of a strain of oral streptococcus could be recovered from the gels after the electrophoresis. The facilitation of the electrophoresis of human mucus glycoprotein's at high temperature may reflect the dissociation of macromolecular aggregates of the mucins under these conditions. A number of purified, non‐mucin proteins also moved as discrete bands at 80degreeC. It is suggested that electrophoresis at elevated temperatures should be considered generally for the analysis of multimolecular systems that are of biological importance.
Ultrasensitive silver‐based color staining of polypeptides in polyacrylamide gels
Authors: Sammons David W. , Adams Lonnie D. , Nishizawa Edwards E.
Published: May 2005

A color development system for staining polypeptides in one‐ and two‐dimensional polyacrylamide gel electrophoresis is described. The basis of the Process involves the complexing of silver with polypeptides reactive centers. The reaction is initiated by placing a polypeptide‐containing gel, previously equilibrated with an appropriate concentration of silver nitrate, into a reducing solution that contains sodium hydroxide, sodium borohydride, and formaldehyde. After an appropriate time in the reducing solution, the gel is equilibrated through two changes of an enhancing solution that contains sodium carbonate. The sodium carbonate is necessary for optimal color appear in the polypeptide ‐silver complexes after several hours in the enhancing solution and are best appreciated while viewing over a fluorescent light box that radiates light at 5000°K. The color of each polypeptide‐silver complex is clearly visible above the light background of the stained polyacrylamide gel. Colors of stained polypeptide are blue, green, yellow, and red. Subtle shades of colors also appear and thereby allow easy discrimination of overlapping spots of polypeptides in a two‐dimensional gel. To illustrate the method's relative sensitively, a two‐dimensional pattern of human fibroblast polypeptides is compared with patterns of a duplicate gel that is stained with Coomassie Blue and developed by autoradiography. The sensitivity of the silver stain process is superior to Coomassie Blue and is comparable to autoradiography after in corporation of conventional levels of 35S‐ methionine. The utility of the procedure for identifying and characterizing human proteins is illustrated by staining human proteins is illustrated by staining human plasma and platelet polypeptides after two‐dimensional gel electrophoresis. The gel electrophoresis color development system consists of steps that are simple, reproducible, and sensitive, and most importantly, which yield colored polypeptide‐silver complexes that are reproducible from gel to gel and tissue to tissue.
The nature of observed schlieren patterns in isoelectric focusing gels and their use for position of banded proteins
Authors: Edwards Jesse J. , Anderson Norman G.
Published: May 2005

A simple and versatile optical technique for viewing discontinuities in the thickness of horizontal isoelectric focusing gels is described. The discontinuities in the gel are reproducible and relate directly to the ampholyte distribution. Characterization of the capture of the discontinuities in the gel is presented as well as a demonstration of how they may used for localization of focused proteins.
Miniature ultrathin‐layer isoelectric focusing in 20–50 μm polyacrylamide gels
Authors: Kinzkofer Angelica , Radola Bertold J.
Published: May 2005

Ultrathin‐layer isoelectric focusing in polyacrylamide gels on silanized supports provided the means for developing a miniature system combining short focusing time with high resolution and operational simplicity. The essence of miniature ultrathin‐layer isoelectric focusing is the combined use of short separation distances (1–3 cm) and high field strengths (400–700 V/ cm), which can be applied in standard equipment simply by reducing the thickness of the gel to 20–50 um for improved heat dissipation. By the criterion of coalescence of proteins migrating from different positions, a steady state is reached in pH 4–9 servalyt T carrier ampholytes on 1 cm gels after 2 min (15 Vh) and on 3 cm gels after 10 min total focusing time (110–130 Vh). On 3 cm gels the resolution is 0.03–0.035 pI in wide‐range carrier ampholytes, and 0.01–0.02 pI in narrow‐range (0.3 pH units) carrier ampholytes isolated by preparative isoelectric focusing of pH 4–9 Servalyt T in Bio‐Gel P‐60 layers. Miniature ultrathin‐layer isoelectric focusing can easily be adapted to the analysis of multiple samples. When the volume is limited to 0.2–0.4 μl, up to four samples per cm can be applied directly on the gel surface. At this loading, the gel volume per focused sample amounts to only 0.5–5 μl. The gel volume per sample is reduced by a factor of 10–200, relative to the 10–12 cm separation distance in 50–100 μm gel layers. This results in a considerable saving of carrier ampholytes and reagents for gel preparation. For serial dilutions of pH marker proteins, the sensitivity of protein detection after staining with Serva Blue G or Serva Violet 49 is 10–15 ng protein in the miniature system. Miniature ultrathin‐layer isoelectric focusing should prove to be an attractive method for routine applications in clinical, industrial and research laboratories in those cases when only small amounts of material are available or when rapid results are required. The method is particularly suitable for studies of genetic polymorphisms of proteins and enzymes.
Generation of stable pH gradients for preparative isoelectric focusing by electrolysis of two‐component buffer solutions in a multi‐compartment apparatus
Authors: Jonsson Mats , Fredriksson Stig
Published: May 2005

A widened use of isoelectric focusing for preparation of large amounts of pure proteins is highly desirable but is severely hampered,inter aliaby the high cost of the carrier ampholytes used for creating the pH gradient. Efforts are therefore being made to replace the carrier ampholytes with simple two‐component buffers. To this end, a multimembrane electrolyzer, consisting of 22 compartments with attached cooling loops, has been constructed. The choice of membranes has proved to be one of the main problems. With permeable membranes, even small differences in hydrostatic pressure between the ends of the electrolyzer will cause internal liquid flows that prohibit the formulation of a useful pH gradient. Likewise, flow‐tight membranes generally give rise to a large internal flow due to electroosmosis. Among the types of membranes so far examined, polycrylamide membranes form an exception. With these membranes, useful and stable pH gradients have been created. There are drawbacks, however, relating to the low mechanical strength and to the difficulty of producing exactly equal membranes, thereby fulfilling the theoretical demand of constant transference numbers for the ions. The experimental observation that even small disturbances, such as variations of the transference numbers or a minute electroosmotic flow through the electrolyzer, may completely destroy the pH gradient, has been related to the mechanism operating during electrolysis of a simple buffer. While a possible local breakdown of a carrier‐ampholyte pH gradient is rapidly repaired by the current, a disturbance occurring in a buffer pH gradient can be repaired only by diffusion, which is a slow process.
Polymerization kinetics of polyacrylamide gels I. Effect of different cross‐linkers
Authors: Gelfi Cecilia , Righetti Pier Giorgio
Published: May 2005

Polymerization kinetics of polyacrylamide gels, cross‐linked with the following: N,N′‐methylenebisacrylamide (Bis), N,N′‐bisacrylylcystamine (BAC), N,N′‐ diallyltartardiamide (DATD), N,N′(1,2‐dihydroxyethylene) bisacrylamide (DHEBA) and ethylene diacrylate (EDA), have been studied. The gels are polymerized directly in a spectrophotometer quartz cuvette and the kinetics followed at 283 nm (disappearance of the double bond) or at 600 nm (Tyndall effect due to turbidity of Bis, DHEBA and BAC gels). The order of reactivity of the various cross‐links appears to be: Bis ≅ DHEBA > EDA ≅ BAC ≫ DATD. The last cross‐link, (DATD), was found to actually be an inhibitor of gel polymerization, leading to highly unpolymerized gels, especially at high %C values. Bis and DHEBA gels, at 3 to 5 %C levels, are fully polymerized within 30 min at room temperature, while EDA and BAC gels, in the same %C range, require at least 3h. All cross‐links, when used above 10 %C, display quite slow polymerization kinetics, requiring overnight reaction for good conversion of monomers into polymer chains. When polymerizing liquid linear polyacrylamide (without cross‐link), an initial concentration of > 5% should be used and the reaction continued overnight, at room temperature or higher, for acceptable polymerization efficiency. On the basis of these data, a new structural model for highly cross‐linked gels is proposed.
The application of direct tissue isoelectric focusing to the study of human skeletal muscle
Authors: Thompson Basil J. , Burghes Arthur H. M. , Dunn Michael J. , Dubowitz Victor
Published: May 2005

A method of direct tissue isoelectric focusing (DTIF) in agarose of human skeletal muscle is described. This particular method was developed to utilize the small amounts of tissue obtained by needle muscle biopsies performed for diagnostic purposes. 20 μm thick cryostat sections were adhered to the hydrophilic surfaces of small GelBond rectangles. These were then applied directly to the surface of the gels. A charge‐balanced purified agarose was used to make the gels, which contained Triton X‐100 to enhance protein solubilization. Stabilization of the pH gradient was attempted by employing a 3% w/v ampholyte which was a blend of 0.85% w/v pH 8–10.4 and 2.15 % w/v pH 3–10 Pharmalyte, by using anolyte regulation with 0.1 M aspartic acid and by focusing the gels under a CO2‐extracted nitrogen atmosphere. Equilibrium of proteins stained by Coomassie Brilliant Blue R‐250 was apparent as monitored by comigration of sections from both anode and cathode. With the exception of the cathode end, excellent resolution and reproducibility was achieved. Better cathode resolution was noted with non‐equilibirium conditions. Preliminary zymograms of lactic dehydrogenase (LDH), using a tetrazolium technique, have shown consistent patterns of multiple isoenzymes, the basic components of which were best seen using non‐equlibirium conditions.
Steady‐state rheoelectrolysis — a method for sioelectric focusing without carrier ampholytes. II. The pH course to be expected on rheoelectrolysis of a buffer solution composed of a weak acid and a weak base
Authors: Rilbe Harry , Stockholm
Published: May 2005

Steady‐state rheoelectrolysis of a salt of a weak base and a weak acid, with a possible excess of base or acid, is treated theoretically. For an accurate deduction of the pH course through the region where the pure salt prevails, the treatment is based directly on the mass action equations and not on Henderson's equation. However, the conductivity contributions of the water ions H+ and OH− are not taken into account, a limitation that restricts the validity of the theory roughly to the pH region between 4 and 10. The pH span that can be covered by this technique is found to be about 2.5 units bigger than the pK difference between base and acid. An exact expression for the pH gradient is deduced, and it is shown that this gradient has a maximum at the location of the pure salt and minima in the neighbourhood of the two pK values. There is no theroretical obstacle to the use of this technique for isoelectric focusing without carrier ampholytes.
Isozyme comparisons using weighted analyses of electrophoretic mobility in a range of polycrylamide gel concentrations
Authors: Fieldes Mary A. , Tyson Hugh
Published: May 2005

Peroxidase isozymes from two flax genotypes were separated electrophoretically using a range of concentration, regression models were fitted by weighted least squares method, using matrix techniques and appropriate programmes, weight were reciprocals of variances of significance, as a chi square, of the residual sums of square (SS). Log (Rm) Plotted against gel concentration showed significant curvilinearity; Log (P/Hb), where P was an isozyme's migration was linear with concentration. Matrix method allowed a completely generalized approach to the analysis of data of this type. In particular, regression data could be readily combined so as to allow orthogonal comparison between Intercepts and slops in an analohgous fashion to the orthogonal breakdown of row, column and interaction effects in a balance two‐way analysis of Weighting effected some improvement in comparison between isozymes; combined analyses of weighted regression produced striking improvement in discrimination relative to simple pairwise Comparisons between isozymes.
Silver Stains for proteins in polyacerylamide gels: A comparison of six methods
Authors: Ochs Denis C. , McConkey Edwin H. , Sammons David W.
Published: May 2005

Six of the most recent silver staining method for detecting polypeptides on polyacrylamide gels were compared. We found the method of Sammons et al. (Electrophoresis, 1981, 2, 135–141) to be least expensive and most reproducible, For staining gels of 1.5 mm in thickness, it is also the most sensitive method. Other methods may be preferable for staining gels 0.8 mm or less in thickness.
Determination of molecular weights and Stokes' radii of non‐denatured proteins by polyacrylamide gradient gel electrophoresis. 1. An equation relating total polymer concentration, the molecular weight of proteins in the range of 104–106, and duration of electrophoresis
Authors: Rothe Gunter M. , Purkhanbaba Huschang
Published: May 2005

Untreated and processed gel plates of polyacrylamide (PAA) gradient gels were cut into strips perpendicularly to their length, and the wet and dry matter of the sections was determined. In untreated gels the apparent dry matter, as well as the relative dry matter, are a linear function of the gel length. In processed gels, however, only the apparent gel concentration increases linearly with the gel length, whereas the relative dry matter increases linearly with the square root of the gel length. The %T (content in polyacrylamide) was calculated from the apparent dry matter. The gel gradients used were found to be linear with respect to %T. Six different calibration proteins were run and their time‐dependent migration distances determined. For each protein a linear relationship exists between the square root of its migration distance (√D) and the logarithm of the electrophoretical duration (log t). In this relation √D can be substituted by the square root of the PAA concentration \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or by the relative dry matter (TM(%)). The resulting straight lines seem to intersect near the zero time of electrophoresis and the starting concentration of the gradient. At any time during electrophoresis (t» O), the square root of the migration distance (√D) of the calibration proteins and the logs of their Stokes' radii (log Rs), or the logs of their molecular weights (log MW), are correlated linearly. In this function, √D may be replaced by either \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {\%{\rm T}} $\end{document} or TM (%). It is shown that this correlation is not subject to time, although the constants are changed during the process of electrophoresis. The slopes of the lines and the duration of the electrophoresis (at a constant voltage) are related by a hyperbolic function. Regression lines obtained for different times during electrophoresis intersect in the range of the starting gel concentration. A more general equation is formulated which states ‐ within certain experimental limitations — the size of proteins, their migration distances, and the time of electrophoresis.
Preparative electrofocusing in agarose slab gels
Authors: Cantarow Walter D. , Saravis Calvin A. , Ives David V. , Zamcheck Norman
Published: May 2005

A rapid preparative isoelectric focusing agarose slab gel technique that should prove to be valuable for the purification and characterization of clinically important macromolecules is described in this report. Up to 120 mg of plasma proteins have been focused in a 9.5 ml volume IsoGel agarose slab gel on a hydrophilic polyester plastic backing (GElBond) in 30 min or less. The agarose was permeable to proteins with molecular mass of 5–50 × 106 daltons and allowed recoveries of from 11–100 % depending on the proteins electrofocused. Proteins whose isoelectric points (pI) differed by 0.07 pH units will be separated by the described technique.
Nucleotide isotachophoretic assay: Method and application for determination of ATP/ADP ratio in several rat tissues
Authors: Pérez José A. , Mateo Fátima , Meléndez‐Hevia Enrique
Published: May 2005

Separation of ATP and ADP was achieved in isotachophoresis by using malonate, inorganic phosphate (Pi), creatine phosphate and lactate as spacers, which isolate these nucleotides from other UV‐absorbing products in rat tissues. By combining isotachophoresis with enzymatic end‐point analysis of several rat tissues, calibration for ATP and ADP was achieved obtaining an operative calibration curve that allows the use of isotachophoresis as the only analytical technique in further assays. ATP and ADP quantities of rat skeletal and smooth muscle, heart, liver, kidney and lung were determined by isotachophoresis. Individual variation was also calculated by analyzing 30 animals in the same control conditions, muscular tissues showing the greatest values for ATP and ADP, whereas kidney and lung show the smaller values. ATP/ADP ratio is discussed as a representative parameter for describing the energy content in biological tissues, there are also experimental reasons which imply that other product concentrations in this parameter not be included. This ATP/ADP ratio is calculated in the rat tissues studied, as well as its individual variation, obtaining the larger values for muscular tissues and the smaller for kidney. A great individual variation is found in kidney, followed by heart and skeletal muscle, and the most constant values are found in liver and smooth muscle. The results obtained in this calibration method for isotachophoresis assay suggest the use of a similar procedure in order to apply this technique for other nucleotide analysis.
Un‐supercoiled agarose with a degree of molecular sieving similar to that of crosslinked polyacrylamide
Authors: Buzás Zsuzsanna , Chrambach Andreas
Published: May 2005

A previous observation by Nochumson et al. [7], showing that agarose after reduction or abolition of its supercoiled structure by hydroxyethylation (SeaPrep 15/45), exhibits a molecular sieving effect similar to polyacrylamide in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was confirmed and quantitated by Ferguson plot analysis in PAGE. SeaPrep 15/45 exhibits an effective fiber radius of 0.8 nm, as compared to 23.8 nm for native agarose and 0.4 nm for 2 % crosslinked polyacrylamide. Its effective pore size (estimated as \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt {{\rm K}_{{\rm R polyacrylamide}} } /\sqrt {{\rm K}_{{\rm R agarose}} } $\end{document}) is 0.88 times that of polyacrylamide 2% crosslinked with N,N'‐methylene bisacrylamide (Bis). Although this is not a practical result as yet, due to the low melting temperature and poor gel strength of SeaPrep 15/45, it does sound the death knell for the use of crosslinked polyacrylamide in macromolecular separations, by demonstrating that a linear polymer capable of forming a gel on mere cooling is equivalent to the product of a laborious and relatively irreproducible free radical polymerization. It remains to find a linear polymer with higher melting point and better mechanical strength and adherence to glass walls than SeaPrep 15/45 to make crosslinked polyacrylamide obsolete.