bluuebunny/crossref_metadata_2025 huggingface dataset:
Digital image analysis of isoelectric focusing patterns
Authors: Thomson Alan J. , Peet Frederick , Sahota Tara S. , Morrison Duncan J. , Ibaraki Anthony , White Eleanor E.
Published: May 2005

In a study of intersterility groups in the genus Armillaria, a scanning stage microscope photometer was used to obtain digitized images of photographs of isoelectric focusing patterns. Absorbance values were then mapped from the metric scale to the pH scale. Absorbance values in specified pH intervals were used as features in multivariate statistical procedures to characterize populations and to assign isolates to populations. Digital image analysis techniques were used to obtain an enhanced image of faint or poorly resolved bands.
Crossed immunoelectrophoretic analysis of split products of the third complement factor (C3d) following in vivo activation of the complement system
Authors: Teisner Børge , Petersen Niels E. , Folkersen Jørgen J. , Hau Jann
Published: May 2005

Split products of the third complement factor expressing D but not C epitopes (C3d) were analysed by crossed immunoelectrophoresis using the intermediate gel technique. Four molecular forms of C3d were identified and designated 1, 2, 3 and 4 according to their electrophoretic migration velocity. Whereas the relative concentration of molecular forms 2 and 3 were increased following acute in vivo activation and nephritic factor (NeF)‐mediated in vitro activation, the relative concentration of molecular form 1 was found to be increased in patients with chronic activation of the complement system. The results obtained by the addition of anti‐C3c antibodies to the first‐dimensional gel suggested that form 4 was a C3d‐like molecule split from the native C3 during electrophoresis. By the addition of anti‐albumin antibodies to the first‐dimensional gel it was demonstrated that molecular form 1 of C3d was a complex between C3d and albumin. The immunoprecipitation patterns of molecular forms 2 and 3 was unaffected by anti‐albumin and anti‐C3c antibodies added to the first‐dimensional gel. These data suggest that molecular forms 2 and 3 of C3d are the primary split products of C3b and these molecules are able to form a stable complex with human albumin.
Cellulose acetate electrophoresis of glycosaminoglycans in the central nervous system
Authors: Bertolotto Antonio , Magrassi Maria L.
Published: May 2005

A new electrophoretic system, able to separate all the known standard glycosaminoglycans, was investigated as a method for the study of glycosaminoglycans extracted from tumours of the central nervous system. Densitometric analysis of standard glycosaminoglycan electrophoresis revealed high sesitivity and good repeatability. The electrophoretic system applied to biological samples based on a comigration of extracted glycosaminoglycans with standards, allowed subfractioning of chondroitinsulfates, dermatan sulfate and herapan sulfate and identifaction of heparan sulfate and heparin directly on the sheet by treatment with nitrous acid. Such characters permit the analysis of glycosaminoglycans present in small amount in biological samples.
How to perform subsequent or ‘double’ immunostaining of two different antigens on a single nitrocellulose blot within one day with and immunoperoxidase technique
Authors: Geysen Johan , De Loof Arnold , Vandesande Frans
Published: May 2005

Defining the properties of antibody preparations has been facilitated by immunoblotting techniques: by ‘double’ immunostaining of blots, however, one can clearly visualize the properties of two (or even more) antibodies on a single blot. We describe a one‐day procedure in which two different antisera, both raised in rabbits, can subsequently be applied to a single nitrocellulose blot of protein samples separated by either native or sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis. In order to visualize antibody activity, a three‐step unlabeled immunoezyme method ‐ the peroxidase anti‐peroxidase technique ‐ has been used for both first and second immunostaining. Discrimination between the results of each of the immunostainings has been achieved by using 3,3′ ‐diaminobenzidine and 4‐chloronaphtol as different peroxidase substrates which yield differently colored precipitates. The problem of method overlapping has been overcome by developing a short but efficient elution procedure for the elution of antibody bound to the blot during the first immunostaining: going from ‘mild’ conditions (suited for incubations with sera) to ‘denaturing’ conditions (low pH, low ionic strength, 20 % dimethylformamide) and vice versa repeatedly does not affect antigens but elutes antibodies completely.
The microheterogeneity components of orosomucoid and the dissociation constants and mobilities of concanavalin A/corosomucoid complexes in crossed affinoimmunoelectrophoresis with free concanavalin A
Authors: Hansen John‐Erik Stig , Lihme Allan , Bøg‐Hansenm Thorkild C.
Published: May 2005

In crossed affinoimmunoelectrophoresis with free concanavalin A (Con A) in the first dimension, four orosomucoid components are normally found in human serum. In this study an optimal range of Con A concentration in the first‐dimension gel was defined. The most retarded, fourth component of orosomucoid was shown to be a result of entrapment of Con A‐binding molecules in the affinity precipitate. The affinities of the second, weakly retarded, component and of the third, strongly retarded, component to a Con A binding site were found to be identical. The mobilities in the first‐dimension electrophoresis of the orosomucoid/Con A complexes were found to be significantly different from each other. Compared to the mobility of the nonretarded, first, component of orosomucoid, the second component had a mobility of 40% and the third component had a mobility of 10%. Differences in the antennary carbohydrate structures on the glycosylation sites of orosomucoid are suggested as an explanation for these observations.
Serum fractionation on immobilized pH gradients with one‐ and two‐dimensional techniques
Authors: Gianazza Elisabetta , Frigerio Adele , Tagliabue Anna , Righetti Pier Giorgio
Published: May 2005

Immobilized pH gradients covering, in presence of 8 Murea, the intervals pH 4–7, pH 4–8 and pH 4–9 were applied tothe analysis of human serum. Both formulations with constant buffering power and with constant ionic strengthd were tried. Results are presented of the isoelectric focusing step and of a further fractionation of the sample by sodium dodecyl sulfate‐ polyacrylamide gel electrophoresis in a two‐dimensional by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in a two‐dimensional system.
Computer analysis of two‐dimensional gels: Automatic matching
Authors: Miller Mark J. , Olson Arthur D. , Thorgeirsson Snorri S.
Published: May 2005

A program which automatically mathches the spot patterns resolved on two‐dimensional gel electrophoretograms is described. The program does not require handmatched, landmark matches for initial alignment of the spot patterns. The matching algorithm is based on a hierarchical nearest neighbor analysis. Starting with the most intense spots in the two films, the program determines if spots are equivalent by attempting to match a list of all nearby spots (a “cluster”) from each film. “Clusters” are considered to be aligned if there is a low probability that the pairing of spots between them is due to a random process. The match is further tested by requiring that secondary clusters (i. e. those clusters that surround the spots matched between the central clusters) can also be aligned. Pairings found by cluster matching are checked for consistency and matches that are out of alignment with the majority of other mathces are eliminated. Finally, the program tries to match the remaining spots by mapping the coordinates of an unmatched spot in one gel into the coordinate system of the other using the matched spots in the cluster as landmarks. The formulas used for the transformation allow for localized rotation and stretching of the coordinate systems. The accuracy and robustness of the program are discussed. If high quality gels are used, the program will find, on average, 95% of the spots that are mathcable between the gels, and requires about 4.3 seconds per mathch. Most of the errors are found at the extreme edges of the gels.
Some problems encountered in the selection of electrolyte systems in isotachophoresis
Authors: Boček Petr , Gebauer Petr
Published: May 2005

A theory is described which gives a new algorithm for the selection of suitable electrolyte systems based on assuming (i) the zone stability as its correct isotachophoretic migration; (ii) the separability as the establishment of a fixed migration order of the zones of individual substances. The theory is based on data of the typeūi,j, i. e., effective mobility of substance i in the j‐th zone. For checking the stability of zones, the use of the zone existence diagrams is demonstrated, where the groups of substances giving stable zones are easily characterized, with special attention to anionic analyses with OH− as the terminator. For checking the separability of substances, the use of the sequence contours in an existence diagram is demonstrated with respect to the zone order as well as to the formation of stable mixed zones. Experiments are given where anionic isotachophoresis of some amino acids with OH− as the terminator is shown, and the formation of stable mixed zones of substances with mutually different effective mobilities is demonstrated.
Enhanced resolution of phosphoglucomutase (PGM1) by the addition of a separator to ultrathin isoelectric focusing gels
Authors: Gill Peter , Sutton John G.
Published: May 2005

Enhanced separation of phosphoglucomutase (PGM1) isoenzymes has been achieved by addition of the separator N‐(2‐Hydroxyethyl)piperazine‐N′‐3‐propanesulphonic acid (EPPS) to pH 5–7 Ampholines in ultrathin and 1 mm isoelectric focusing gels. The resultant pH gradient ranges between pH 5.6 to pH 5.9 and effectively spreads PGM1 isoenzymes across the whole width of the gel. The technique has been shown to be especially useful for separating some of the rare PGM1 variants. The use of different electrolyte combinations has also been investigated and it has been shown that strong electrolytes, e. g. sodium hydroxide and orthophosphoric acid, result in a small but statistically significant increase in the gradient range compared with weaker electrolytes such as threonine/histidine. In addition, the effect of temperature on the rate of migration of PGM1 isoenzymes has been investigated.
Electrophoresis of dimethylcyanamidated mercaptoacids
Authors: Wroński Mieczyslaw
Published: May 2005

The conversion of a sulfydryl group of mercaptoacids to a thiuronium group by means of dimethylcynamide followed by electrophoresis on cellulose gel strips is suggested as a method for estimation of the number of carboxyl groups in the molecule and for separation of mono and polycarboxyl mercaptoacids. As a result of cyanamidation the molecule gains an additional positive charge reflected in a shift of pH‐mobility curves along the mobility axes.
Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining
Authors: Heukeshoven Jochen , Dernick Rudolf
Published: May 2005

The study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1–0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides of molecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS‐containing gels and a simple spot test on thin gellayers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.
Isoelectric focusing of monoclonal immunoglobulin G, A and M followed by detection with the avidin‐biotin system
Authors: Chiodi Francesca , Sidén Åke , Ösby Eva
Published: May 2005

The sera of 49 subjects with IgG, IgA or IgM benign monoclonal gammopathy‐components were examined by agarose gel isoelectric focusing followed by Coomassie Brilliant Blue R‐250 staining as well as avidin‐biotin amplified double‐antibody peroxidase labelling after nitrocellulose blotting. IgG‐components gave 2–10 distinct bands within the isoelectric point (pI)‐range of pH 6.5–9.5, IgA‐components were focused into 10–15 bands with pI‐values of pH 4.5–6.5 and the IgM‐components gave 1–2 fractions within the pI‐range of pH 4.5–6.5. The avidin‐biotin peroxidase complex (ABC) technique exhibited some tendency to non‐specific labeling related to the avidin‐biotin component and depending on the relative amounts of the individual proteins. However, the method has a definite potential for the visualization of submicrogram quantities of immunoglobulins blotted to nitrocellulose membranes after isoelectric focusing.
The use of zwitterionic buffers in the separation of vaginal and seminal acid phosphatases in isoelectric focusing gels and the effect of neuraminidase treatment
Authors: Gill Peter , Sutton John G.
Published: May 2005

Seminal acid phosphatase (SAP) and vaginal acid phosphatase (VAP) bands, visualised by conventional isoelectric focusing have a wide pH range extending from approximately pH 4.0–6.0. Although a large proportion of these bands had similar isoelectric point (pI) values, the extremities of their respective ranges do not overlap. For SAP it was found that coincidental focusing tended not to occur withion a pH range of approxiamtely 4.3–5.2. The introduction of zwitterionic buffers to a conventional pH 4.0–6.0 Ampholine gradient produces a narrow pH gradient (4.3–5.2) in which semen‐specific SAP bands can be distinguished from VAP. This system has been used in an attempt to qualitatively identify SAP in the presence of VAP from a number of semen‐contaminated swabs. The interconvertibility of SAP into VAP has been demonstrated by neuraminidase treatment of SAP. When SAP was visualised on 15 cm gels containing pH 4–6 Ampholines it was observed that anodal bands faded and were converted into cathodal forms. Detailed examination of cathodal VAP and neuraminidase‐treated SAP was carried out on narrow pH gradients which enabled direct comparisons of individual isoenzymes. This provided confirmation that interconversion between the two forms was possible. Finally, a population survey of SAP patterns was carried out which failed to reveal any genetic polymorphism.
Identification of a so‐far not characterized human serum protein associated with essential hypertension
Authors: John Andrea , Henke Jürgen , Morich Frank J.
Published: May 2005

According to recent observations the plasma of hypertensive humans contains an additional protein which can be detected by one‐dimensional sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. A particular function of this protein, called “hypertension associated protein” (HAP), has not yet been defined. In this study the correlation between the appearance of this protein and hypertension was only weak in a group of 100 persons. However, by comparing the presence or absence of this protein as detected by polyacrylamide gel electrophoresis with the results of haptoglobin typing of the same plasma samples, one finds that this protein is the alpha‐1‐chain of haptoglobin. Persons with HAP in their plasma belong either to the Hp (1) or to the Hp (2–1) type. Thus the determination of haptoglobin phenotype is possible by one‐dimensional SDS‐polyacrylamide gel electrophoresis, which might be of importance for forensic serogenetics. Genetically controlled phenotypes of haptoglobin are known to be associated with a higher risk for different diseases. According to the results of this study hypertension is also among these diseases.
Hybridisoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes: Demonstration of human globins
Authors: Altland Klaus , Rossmann Ute
Published: May 2005

In contrast to hitherto described procedures, gels containing immobilized pH gradients were prepared from buffered solutions of constant, near neutral pH at ambient temperature. The washed, dried and rehydrated gels were run in the presence or absence of free carrier ampholytes, 8 M urea, Triton X‐100, 2‐mercaptoethanol and dithiothreitol, respectively. The patterns of human globins were compared for linear and modified pH gradients in the range between pH 6 and 10. By adding free carrier ampholytes to the gels the prerun time of urea‐containing gels was reduced from many hours to 60–90 min. The run time with samples was reduced from overnight to 2 h at 3000 V per 10 cm. The amount of sample which had to be applied to obtain clearly visible patterns was found to be close to that used for conventional isoelectric focusing with carrier ampholytes. Upon drying in the laboratory atmosphere an oxidizing activity is generated in polyacrylamide gels which produces artifacts of selected test proteins when separated in the rehydrated gels. The addition of 2% 2‐ mercaptoethanol or 50 mM dithiothreitol to the rehydration solution prevented the formation of these artifacts. Hybrid isoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes combines the advantageous properties of isoelectric focusing in immobilized and conventional carrier ampholyte generated pH gradients.
The quantitation of proteins in silver stained polyacrylamide gels
Authors: Yüksel K. Ümit , Gracy Robert W.
Published: May 2005

This study investigates some of the problems encountered in quantitation of proteins in silver stained polyacrylamide gels. Subunit molecular weight, isoelectric point and amino acid content are examined as possible factors for differential staining intensities of proteins. We also describe a procedure for quantitation using atomic absorption spectroscopy, independent of the colors formed upon silver staining. Using this method we show that some proteins such as pepsin bind silver but fail to develop visual color reactions.
Gene frequency distribution of the B subunit of factor XIII (F XIIIB) in Minnesota Whites, Blacks and Amerindians
Authors: Miller Shirley A. , Dykes Dale D. , Polesky Herbert F.
Published: May 2005

Serum specimens from local Whites, Blacks and Amerindians were phenotyped for the B subunit of Factor XIII. Isoelectric focusing of desialylated samples on agarose gels at a pH 4–7 gradient followed by an immunoblotting technique was used for band identification. Gene frequencies for the three common alleles in Whites, F XIII*1, F XIIIB*2 and F XIIIB*3 were 0.776, 0.088 and 0.136, respectively, and were consistent with reports on this genetic marker system in European Whites. The Blacks and Amerindians were clearly differentiated from the Whites with F XIIIB*1,2 and 3 frequencies of 0.286, 0.635 and 0.079, respectively, for Blacks and 0.500, 0.034 and 0.466 for Amerindians. Based on the available population data on Factor XIIIB, this genetic system should prove to be a valuable marker for anthropological genetics and parentage testing.
Protein staining of 6 mm diameter sodium dodecyl sulfate‐polyacrylamide gels within one and a half hours
Authors: An der Lan Birgit C. , Auzan Colette , Sullivan James V. , Chrambach Andreas
Published: May 2005

A device for rapid rotation of perforated gel holding tubes has been constructed by which the diffusion of sodium dodecyl sulfate (SDS) from cylindrical polyacrylamide gels can be substantially accelerated. This device allows one to stain protein bands obtained in SDS‐polyacrylamide gel electrophoresis (9 % gel concentration) within one and a half hours, using the procedure of fixation in trichloroacetic acid followed by Coomassie Brilliant Blue G‐250 staining, which requires no destaining.
Membrane protein analysis by isoelectric focusing in immobilized pH gradients
Authors: Rimpilainen Marja A. , Righetti Pier Giorgio
Published: May 2005

Non‐ionic detergents (NP‐40, Triton X‐100) have poor solubilizing properties on membrane proteins, as demonstrated by separating NP‐40‐extracted proteins by isoelectric focusing also in the absence of carrier ampholytes (Ampholine), i. e. in immobilized pH gradients (IPG). Addition of 4% Ampholine to the sample and to the IPG gels results in an increase in membrane solubility, from 40% in NP‐40 alone to 60% in NP‐40 with 4% Ampholine, when both extractions were in the absence of 8 M urea. If the latter solvent is used, these values are increased to 85% for the former and to 100% for the latter extraction conditions. In presence of carrier ampholytes sharp bands are obtained on isoelectric focusing. It is hypothesized that neutral detergents in presence of Ampholine form mixed micelles, mimicking the properties of natural zwitterionic surfactants.
Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis
Authors: Neuhoff Volker , Stamm Reinhard , Eibl Hansjörg
Published: May 2005

A systematic analysis of protein staining in polyacrylamide gels with Coomassie Brilliant Blue (CBB) R‐250 and G‐250 using a high resolution densitometer allowing for quantitative measurements during staining and destaining has revealed that none of the published procedures allows quantitative measurements. Protein staining with CBB R‐250 in methanol/water/acetic acid is poor, as is staining with CBB G‐250 in trichloroacetic acid or perchloric acid, the latter two, however, allowing for a weak background staining. Consequently using the colloidal properties of the CBB dyes, stronger for G‐250 than for R‐250, it is possible to increase the sensitivity of protein staining to a detection limit of 0.7 ng bovine serum albumin/mm2 gel. In addition, sensitive protein staining on a clear background is possible. Recipes are described (Section 3.11) for intensified protein staining with CBB G‐250 using trichloroacetic acid or perchloric acid on a clear background. Optimal staining of proteins on a clear background can be performed with phosphoric acid and CBB G‐250 in the presence of ammonium sulfate since under these conditions the colloidal state of the dye is optimized. Furthermore, conditions are described which allow the stable fixation of the protein‐dye complex. Combining the optimized staining conditions with the stable fixation in 20% ammonium sulfate allows for stepwise staining for e. g. detection of weak spots in addition to intense protein spots. The dependence of different staining procedures on gel thickness, gel concentration and compounds routinely used in polyacrylamide gel electrophoresis is also analysed. Calibration curves and application of the new procedure to biological material demonstrate its wide applicability. Convincing arguments for the colloidal properties of the CBB dyes are presented, formulating the rationale for intensified protein staining with CBB dyes in polyacrylamide gels without background staining.
Silver staining of immunofixed group specific component on cellulose acetate membranes after isoelectric focusing in narrow pH interval gels
Authors: Westwood Sara A.
Published: May 2005

The sensitivity of group specific component (Gc) detection by immunofixation after isoelectric focusing (IEF) has been examined by using two IEF methods: ultrathinlayer IEF and immobilized pH gradient (IPG) focusing followed by two staining methods: Ponceau S and a combination of Ponceau S with silver staining (SS). Both diluted plasma and bloodstain extracts were examined by these methods at two dilutions of immunofixation antibody ‐ 1:6 and 1:24. The cellulose acetate membranes containing the Gc precipitates were washed with sodium chloride after the SS process. This greatly improved the visibility of the Gc bands. The method was found to be more sensitive than Ponceau S and its use reduced the cost of immunofixation by 75%. Ultrathin‐layer IEF was carried out in gels containing the pH 4.5–5.4 Pharmalytes. The separator N‐2‐hydroxyethylpiperazine‐N′ 2‐ethanesulfonic acid (HEPES) was added to some gels and the effect on the resolution of the Gc 1 subtypes was examined and compared with the separation achieved with IPG gels. It was found that ultrathin‐layer IEF using narrow range Pharmalytes and the separator HEPES was the more sensitive method; however, IPG gels were easier to interpret.
Reliable phenotyping of esterase D by low voltage isoelectric focusing: Evidence for the new variant ESD Yamaguchi
Authors: Yuasa Isao , Tamaki Nobuto , Suenaga Kazuyuki , Ito Keiichi , Inoue Terutaka , Okada Kichiro
Published: May 2005

Low voltage isoelectric focusing (IEF) for the classification of esterase D (ESD) has been developed. This new phenotyping method is based on non‐equilibrium IEF. The obtained patterns might be isotachophoretic because the ESD bands were concentrated sharply and the values of their isoelectric point (pI) were somewhat higher than those found by equilibrium IEF. The method proved to be fast, easy and reliable for the differentiation of the six phenotypes determined by three codominant alleles, ESD*1, ESD*2 and ESD*7. The distribution of ESD phenotypes in Western Japan has been investigated and a new rare variant, ESD Yamaguchi (ESD Y), characterized by a slightly cathodal mobility to ESD 2, could be identified. The frequencies for the three alleles ESD*1, ESD*2 and ESD*7 were 0.600, 0.389 and 0.011, respectively.
Silanization of supporting glass plates avoiding fixation of polyacrylamide gels to glass cover plates
Authors: Tegelström HÅKan , Wyöni Per‐Ivan
Published: May 2005

A common problem, when using Silane A 174 to bind polyacrylamide gels to supporting glass plates, is that the gel also binds to the cover glass in the gel casting frame. This can be avoided by using a low concentration of Silane A 174 and by rinsing the supporting glass plate with acetone after the Silane A 174 treatment.
Electrophoretic migration velocity of amphiphilic proteins increases with decreasing Triton X‐100 concentration: A new characteristic for their identification
Authors: Børsum Tone , Bjerrum Ole J.
Published: May 2005

The relation between the electrophoretic migration velocity and the concentration of the non‐ionic detergent Triton X‐100 in the buffer was studied by crossed immunoelectrophoresis of hydrophilic and amphiphilic proteins. The migration velocity of hydrophilic human plasma proteins was not affected by the presence of Triton X‐100 in the agarose gels in the concentration range of 1–0 % v/v. In contrast, amphiphilic proteins, like the human erythrocyte proteins glycophorin, band 3 protein and acetylcholinesterase, and the plasma high density lipoprotein (HDL) apolipoprotein, showed increasing migration velocity with decreasing detergent concentration in the gels. An increase of 20–80 % was observed for different amphiphilic proteins when the Triton X‐100 concentration was lowered from 1 % to 0.03 % v/v. This probably reflects the influence of the size of the bound detergent micelle on the charge density of the protein. Thus, by performing corssed immunoelectrophoresis with different concentrations of Triton X‐100 in the first‐dimensional gel it is possible to identify amphiphilic proteins. Presence of 1 % v/v Triton X‐100 in the second‐dimensional gel ensures reliable identification of the precipitates.
Electrophoresis on uncrosslinked polyacrylamide: Molecular sieving and its potential applications
Authors: Tietz Dietmar , Gottlieb Melvin H. , Fawcett John S. , Chrambach Andreas
Published: May 2005

Uncrosslinked polyacrylamide, a viscous liquid, provides a molecular sieving effect in electrophoresis qualitatively the same as that of crosslinked gels. This finding confirms previous results by others using uncrosslinked polyacrylamide mixed with various gel matrices. It provides a sieving medium constituted by an entirely and necessarily random association of inert fibers, as stipulated for interpretation of migration by the Ogston theory. Ferguson plots of several proteins in uncrosslinked polyacrylamide exhibit Yo values smaller and less divergent from one another than in 5 % crosslinked gels, suggesting a curvature of the Ferguson plots of crosslinked polyacrylamide gels at low %T. If linearity of these plots at low %T, 0 %C could be demonstrated, a means would be found to determine accurate values of free mobility by their linear extrapolation to 0 gel concentration. Potentially, uncrosslinked polyacrylamide at low concentrations may provide a tool for the analytical sieving of subcellular particles. However, electrophoresis in uncrosslinked polyacrylamide at concentrations of less than 10 % is perturbed by convection, and thus these potential applications will depend on combining it with a suitable anticonvective device.
A device for preparative elution electrophoresis using a polyacrylamide gel slab
Authors: Carpenter Helen C. , Skerritt John H. , Wrigley Colin W. , Margolis Joel
Published: May 2005

A compact device, capable of being clipped onto the end of a 3 mm thick analytical polyacrylamide slab gel, has been developed for the collection of resolved protein zones as they elute from the end of the gel. Its unique feature is that eluted proteins were collected between two polyacrylamide‐embedded paper membranes and removed either by continuous or intermittent buffer elution. Because of this direct coupling to large gels identical to those used for analytical electrophoresis, protein zones were obtained with high resolution. The method was applicable to non‐denaturing cathodic and anodic electrophoresis, as well as sodium dodecyl sulfate electrophoresis. Medium‐scale quantities (up to 10 mg/cm2 cross‐sectional area) of protein were fractionated in as little as 4 h with high recoveries.
Fast silver staining of polyacrylamide gels at elevated temperatures
Authors: Biel Hans J. , Gronski Peter , Seiler Fritz R.
Published: May 2005

Renouncing protein fixation with aldehydes, the polyacrylamide gels are incubated in alcoholic solutions of silver nitrate containing acetic acid at 60–70 °C. for a short time and developed with NaOH, also at 60–70 °C. Using various methods of protein fixation, the detection limits of human albumin were determined for 0.75 and 1.5 mm thick polyacrylamide gels with appropriate thickness correction. An overlay technique with silver nitrate containing polyacrylamide gels is described. Dispersing Coomassie Brilliant Blue G‐250 in a solution containing HNO3 and NH4NO3 allows rapid silver staining of prestained gels.
Mathematical simulation of isotachophoresis boundary between protein and weak acid
Authors: Shimao Kazuo
Published: May 2005

Mathematical simulation of an isotachophoretic boundary between protein and weak acid was made on the assumption of steady state. In order to deal with a protein molecule, a Gaussian model for charge distribution of protein molecule was constructed, based on the assumption that acidic and basic residues on a protein molecule dissociate independently of each other. The results were roughly in agreement with protein stacking experiments with regard to boundary thickness and concentration of the stacked zone.
Electrophoretic characterisation of human parotid saliva protein fractions isolated by preparative isoelectric focusing
Authors: Hallinan Frank M. , Rose Marie , Eagleton Marie , Tempany Edward
Published: May 2005

Human parotid saliva proteins fractionated by preparative isoelectric focusing into 3 main fractions have been characterised electrophoretically and immunochemically. The anodic proteins constitute a polymorphic family of collagenase and trypsin sensitive, immunochemically related, phosphoproteins. Many of these contain a common polypeptide of Mr 23 000 either alone or in combination with other lower (Mr 21 000) or higher (Mr 25 000) molecular weight polypeptides. Parotid amylase was substantially purified by this technique and an antiserum against these fractions reacted monospecifically with amylase in a number of systems. The cathodic proteins are all collagenase sensitive proteins with Mr values of 12 500 to 77 000.
Comparison between agarose gel electrophoresis (Panagel) of cerebrospinal fluid with silver staining, and polyacrylamide disc gel electrophoresis for demonstration of the oligoclonal pattern in neurological disorders
Authors: Plan Elisabeth , Seneterre Jean‐Bernard , Caudie Christiane , Quincy Claude
Published: May 2005

A high resolution agarose gel system (Panagel) with silver staining and polyacrylamide disc gel electrophoresis were used to demonstrate oligoclonal bands in cerebrospinal fluid (CSF) from 150 patients identified as having either inflammatory neurological diseases (110 patients) or non‐inflammatory neurological diseases (40 patients). The detection of oligoclonal band patterns in high resolution agarose electrophoresis is sensitive: 65% (polyacrylamide gel electrophoresis: 50%) and specific: 95% (polyacrylamide gel electrophoresis: 73%) with respect to inflammatory neurological diseases. This procedure is easier to perform than polyacrylamide disc gel electrophoresis and is recommended for routine examination of CSF especially for the detection of oligoclonal bands.
Involvement of carbohydrate moieties in the heterogeneities of human blood clotting factor IX
Authors: Polack Benoǐt , Freyssinet Jean‐Marie
Published: May 2005

Purified human blood clotting factor IX, although homogeneous by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, showed two peaks when analyzed by crossed immunoelectrophoresis in the presence of calcium. Differential affinity of lectin from wheat germ (WGA) for the two forms of factor IX, detected by crossed affino‐immunoelectrophoresis, demonstrated that carbohydrate moieties are involved in factor IX heterogeneities. The slow migrating peak has a higher affinity for WGA (Kd= 5.26 × 10−7 M) than the fast migrating peak (Kd= 1.29 × 10−5 M). This study emphasizes the usefulness of lectins as tools to assess homogeneity and integrity of glycoproteins.
On‐line fiber optic UV detection cell and conductivity cell for capillary zone electrophoresis
Authors: Foret František , Deml Mirko , Kahle Vladislay , Boček Petr
Published: May 2005

Simple detection cells for on‐line UV absorbance and conductivity detection in capillary zone electrophoresis are described. The UV detection system employs a commercial photometer and optical fibers which are in direct contact with the outer walls of the separation capillary and conduct the light beam perpendicularly across the migrating zones. The conductivity detection cell consists of platinum wires protruding the wall of the separation capillary and thus in direct contact with the electrolytes inside. The electronic system is adopted from a commercially available conductivity detector for isotachophoresis.
Isoelectric spectrum and titration curves of Aleutian disease virus proteins
Authors: Kierek‐Jaszczuk Danuta , Kaaden Oskar‐Rüger
Published: May 2005

Three major virus‐specific proteins (Mr = 85 000, 75 000 and 25 000) were detected in purified preparations of the cell culture‐adapted Aleutian disease virus (ADV) SL3 isolate according to their molecular weight, isoelectric point and titration curve, For all structural proteins, amino acid sequence homologies were demonstrated by proteolytic peptide mapping. The virus‐induced protein Mr 75 000, detected only in lysates of infected cells, revealed a different proteolytic peptide map. It was shown that each of the structural proteins, although demonstrated to be homogeneous in molecular weight, consists of multiple species differing in their isoelectric points. Protein modifications are discussed as possible cause for the charge variations of ADV structural proteins.
Advantages of enzyme‐immuno‐assay after blotting in bloodstain grouping: Application to the haptoglobin groups
Authors: Hoste Bernadette
Published: May 2005

Enzyme‐immuno‐assay after Western blotting with anti‐haptoglobin and anti‐immunoglobulin conjugated‐peroxidase or‐alkaline phosphatase increases the sensitivity of haptoglobin visualization. The detection limit in serum could be improved from 1:8 to 1:256. This method was applied to bloodstains with improved results.
Immobilized pH gradients in capillary tubes and two‐dimensional gel electrophoresis
Authors: Hochstrasser Denis , Augsburger Valérie , Funk Matthieu , Appel Ron , Pellegrini Christian , Muller Alex F.
Published: May 2005

Isoelectric focusing in mixed carrier ampholyte‐immobilized pH gradients (CA‐IPG) is an effective way to separate proteins by charge. A method to prepare CA‐IPG in capillary tubes and the description of the equipment used are outlined. Two‐dimensional gel electrophoresis patterns of human serum and liver biopsies, with isoelectric focusing and CA‐IPG as the first dimension, are presented. The comparison with two‐dimensional gel electrophoresis patterns obtained by conventional carrier ampholyte pH gradient separation shows the excellent resolution and potential of this technique.
Separation by hybrid isoelectric focusing of normal human plasma transthyretin (prealbumin) and a variant with a methionine for valine substitution associated with familial amyloidotic polyneuropathy
Authors: Altland Klaus , Banzhoff Angelika
Published: May 2005

The human plasma transthyretin (TTR, prealbumin) variant TTR(Met30) has been separated from normal TTR(Val30) by double one‐dimensional electrophoresis using polyacrylamide gel electrophoresis followed by hybrid isoelectric focusing. The procedure appears to be appropriate for identification by the screening of patients with familial amyloidotic polyneuropathy of Portuguese type I. This is the second example to support the recently expressed hypothesis (Altland et al., Electrophoresis 1986, 7, 251–259) that substitutions of electrically neutral amino acids (e. g. methionine for valine at position 30) in a polypeptide chain (e. g. the TTR monomer) may be detected by isoelectric focusing under denaturing conditions (e. g. hybrid isoelectric focusing in the presence of 8 M urea, 50 mM dithiothreitol) when the exchange is close to a charged amino acid (e. g. histidine at position 31, pI (TTR) = 5.7, pKa (His) = 6.0).
Elimination of point streaking on silver stained two‐dimensional gels by addition of iodoacetamide to the equilibration buffer
Authors: Görg Angelika , Postel Wilhelm , Weser Johann , Günther Siegfried , Strahler John R. , Hanash Samir M. , Somerlot Luke
Published: May 2005

Silver staining artifacts, unrelated to protein, such as horizontal lines and vertical micro or point streaking on two‐dimensional gels with carrier ampholytes or immobilized pH gradients in the first dimension are efficiently eliminated by adding iodoacetamide to the equilibration buffer of the first‐dimensional gel.
Isoelectric focusing‐affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses
Authors: Hamilton Robert G. , Roebber Marianne , Reimer Charles B. , Rodkey L. Scott
Published: May 2005

Isoelectric point (pI) microheterogeneity, immunoreactivity and specificity of murine monoclonal anti‐human IgG subclass antisera (MoAb) were evaluated in parallel by isoelectric focusing (IEF)‐affinity immunoblotting and enzyme‐linked immunosorbent assay (ELISA). Ascites protein bands in the pH 3 to 9 region of Coomassie Blue‐stained IEF‐polyacrylamide gels corresponded to mouse host proteins such as albumin, transferrin and immunoglobulins. Bands in the pH 5.5 to 8.0 region were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated anti‐mouse IgG. Use of IgG myeloma (antigen)‐coated nitrocellulose in the IEF‐affinity immunoblot allowed detection of the charge micro‐heterogeneity of the MoAbs. As a group, the MoAbs had pIs ranging from 6.1 to 7.8 with a 0.1 to 0.6 pH unit spread. There were 1 to 5 major dense bands flanked by up to 4 minor fainter bands. The pI values and banding patterns as determined by IEF‐affinity immunoblot analysis were consistent both within and between blots. Semi‐quantitative estimates of binding specificity in the IEF‐affinity blot compared well with cross‐reactivity data obtained from a quantitative ELISA. IEF‐affinity immunoblotting is a useful analytical tool for defining the microheterogeneity of monoclonal antibody pI's and for monitoring antibody specificity. Both parameters are indicators of consistency of antibody produced in culture or in ascites by hybridomas which have been repeatedly frozen and thawed over several years.
A voltage gradient‐induced arrest of circular DNA during agarose gel electrophoresis
Authors: Serwer Philip , Hayes Shirley J.
Published: May 2005

During the gel electrophoresis of double‐stranded DNA, interaction with the gel fractionates the DNA by molecular weight and conformation. Here, it is shown that open circular bacteriophage λ DNA (Mr 32.1 × 106) migrates into a 0.2% agarose gel during a first electrophoresis at 0.34 V/cm, but does not migrate when the voltage gradient is subsequently raised to 6.0 V/cm for a second, orthogonal electrophoresis. Linear DNA of the same molecular weight does migrate at 6.0 V/cm. The data suggest that the arrest observed at 6.0 V/cm is caused by threading of circular DNA by gel fibers. These observations have produced a procedure of two‐dimensional agarose gel electrophoresis capable of detecting comparatively small amounts of circular DNA. The concept of threading is potentially useful for understanding of DNA fractionations obtained with pulsed electrical fields.
Use of secondary antibodies for visualization of bound primary reagents in blotting procedures
Authors: Ramlau Jakob
Published: May 2005

Secondary antibodies are used for visualization of bound primary ligands in blotting procedures in several formats. The term “secondary antibody” usually denotes an anti‐immunoglobulin from a species different from the one producing the primary antibody. This secondary antibody is modified so as to produce a visible signal by conjugation with enzyme, fluorophore, radioisotope or suitable particulatematerial. The characteristics of these antibodies will influence both sensitivity and specificity of the assays in which they are used. Thorough knowledge of these characteristics is thus needed for critical evaluation of results. This paper discusses the production and reactivity of secondary antibodies, their immunochemistry and their possible side reactions. Alternative secondary reagents are also discussed with respect to specificity and sensitivity.
Enzymoblotting: Visualization of electrophoretically separated enzymes on nitrocellulose membranes using specific substrates
Authors: Ohlsson Bertil G. , Weström Björn R. , Karlsson Börje W.
Published: May 2005

Enzymoblotting is a technique for the detection of electrophoretically separated enzymes after transfer to nitrocellulose (NC) membranes and subsequent incubation with suitable substrates. After agarose gel electrophoresis or isoelectric focusing in ultrathin polyacrylamide gels, blotting of the separated proteins/enzymes to NC membranes was performed by capillary forces for 30 min. Hydrolases, e. g. pancreatic proteinases, were visualized using amino acid/peptide substrates based either on the β‐naphthol or para‐nitroanilide (pNA) group. Both types of substrates were used with good results, but the pNA substrates provide greater specificity and sensitivity. An oxidoreductase, lactate dehydrogenase, was visualized using an electron transfer dye staining method. Enzymoblotting also allows detection of inactive proenzymes since conversion to active enzymes in situ on the NC membrane may be performed by pre‐incubating the NC membranes with activation enzymes. The enzymoblotting technique offers several advantages over traditional gel staining methods. The enzymes are immobilized on the NC membrane, preventing diffusion during substrate incubation, and as a result, high resolution is retained even after long incubation times; the product obtained as a result of the enzyme activity can be chemically derivatized and bound to the membrane, thus widening the categories of possible substrates; subsequent incubation of the same NC membrane in various substrate solutions can be performed; enzymoblotting can be used in combination with other techniques, e. g., immunoblotting for parallel structural and functional studies.
Separation of red cell membrane proteins by urea‐Triton‐polyacrylamide gel electrophoresis in one‐ and two‐dimensional systems
Authors: Rouyer‐Fessard Philippe , Lecomte Marie‐Christine , Boivin Pierre , Beuzard Yves
Published: May 2005

Urea‐Triton polyacrylamide gel electrophoresis (UT‐PAGE) has been used to separate, in one dimension, membrane proteins present in red blood cells and hemoglobin chains associated with cell ghosts. The membrane proteins are especially well resolved in the high molecular weight range. The protein fractions were further characterized by two‐dimensional gel electrophoresis with UT‐PAGE in the first dimension followed by sodium dodecyl sulfate (SDS)‐PAGE and phosphorylation in the presence or absence of cAMP. A good recovery of spectrin and of band III was obtained in the two‐dimensional system. The proportion of α, β, Gγ and Aγ globin chains can be determined in comparison to the membrane proteins.
A simple, inexpensive method for the salt‐free concentration of proteins for analysis by two‐dimensional gel electrophoresis
Authors: Maresh Grace A. , Dunbar Bonnie S.
Published: May 2005

A rapid, inexpensive method for the salt‐free concentration of small quantities of proteins for analysis by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D‐PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells cultured in vitro.
Cryoglobulinemia: Analysis of isotype, idiotype and antibody activity by composite gel electrophoresis and immunoblotting
Authors: Chu Jia‐Li , Gharavi Azzudin E. , Elkon Keith B.
Published: May 2005

Improved methods for high resolution composite gel electrophoresis under dissociating conditions and electrophoretic transfer of immunoglobulins (Mr 150 000‐1 800 000) to nitrocellulose have been developed. Using these techniques and highly specific antisera to detect either light or heavy chains on nitrocellulose transfers, the immunoglobulin and clonal composition of washed cryoglobulins (3 μg each) could be determined within two days. The results were confirmed by isoelectric focusing and immunoblotting of selected samples. In addition, the method was used for detection of andiotypic determinant and rheumatoid factor activity within components of the cryoglobulins.
Hydrophobic interaction electrophoresis under high hydrostatic pressure: Study of the effects of pressure upon the interaction of serum albumin with a long‐chain aliphatic ligand
Authors: Masson Patrick , Reybaud Joël
Published: May 2005

Hydrophobic affinity electrophoresis under high hydrostatic pressure has been developed to study the interaction between fatty acid‐free bovine serum albumin and a long‐chain aliphatic ligand physically immobilized within the gel matrix. From apparent association constants at various pressures and temperatures, apparent thermodynamic parameters including the volume change in binding were calculated. The results are as expected for hydrophobic interactions between the long‐chain alkyl ligand and a high‐affinity long‐chain fatty acid binding site. The feasibility of high‐pressure affinity electrophoresis is demonstrated. This new high‐pressure technique provides a direct means for studying quantitatively the effects of pressure upon protein‐ligand interactions. It could become a suitable tool for the investigation of protein binding sites' topography.