bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractDefining 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

AbstractIn 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

AbstractImmobilized 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

AbstractA 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

AbstractA 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

AbstractEnhanced 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractSeminal 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

AbstractAccording 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

AbstractIn 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

AbstractThis 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

AbstractSerum 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

AbstractA 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

AbstractNon‐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

AbstractA 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

AbstractThe 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

AbstractLow 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

AbstractA 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.