bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isoelectric focusing of α1‐antitrypsin (PI) using restricted pH‐range carrier ampholytes in combination with a highly cross‐linked gel and a separator
Authors: Klasen Eduard C. , Rigutti Adriana 
Published: May 2005

AbstractA simple technique for polyacrylamide gel isoelectric focusing of α1‐antitrypsin (PI) using thin, highly cross‐linked gels, restricted pH‐range carrier ampholytes and a separator was developed. This method enables easy typing of all known PI phenotypes including the four PI*M subtypes.
Isoelectric focusing studies of antibody clonotype patterns in outbred rabbits during prolonged immunization
Authors: Binion Steven B. , Rodkey L. Scott 
Published: May 2005

AbstractIsoelectric focusing was used to study the antibody clonotype patterns of outbred New Zealand White rabbits during pronlonged immunization with the gram positive bacterium Micrococcus lysodeikticus. Antisera were examined by analytical isoelectric focusing in 1% agarose gels using inexpensive carrier ampholytes which were synthesized in the laboratory. Clonotype patterns of antimicrococcal antibodies were visualized by radioautography using [125I]‐labeled micrococcal carbohydrate antigen. Examination of the antibody clonotype patterns in antisera from a number of outbred rabbits revealed that variations in clonotype expression often occurred within an individual during successive rounds of immunization. These studies demonstrate the utility of analytical isoelectric focusing for monitoring antibody clonotype changes within an individual during immunization.
2‐D Electrophoresis gel data base analysis: Aspects of data structures and search strategies in GELLAB
Authors: Lemkin Peter F. , Lipkin Lewis E. 
Published: May 2005

AbstractSearch strategies for finding spot differences among multiple two‐dimensional (2‐D) polyacrylamide electrophoresis gels are discussed in the context of the GELLAB spot data base management system. A 2‐D gel experiment should have a well‐defined biological experimental and preparation protocol reflecting the hypotheses of the problem. So too should the analysis of its corresponding 2‐D gel computer spot data base have a protocol. This protocol is heavily influenced by the nature of the biological experiment as well as 2‐D gel preparation considerations including the realities of artifactual and systematic noise. It is further influenced by constraints due to computational considerations. The search strategy is that part of the analysis protocol in which an experimenter iteratively defines tests to find significant spot differences. One goal of designing a well thought out search protocol is to reduce the number of search iterations required. Aspects of some requirements and constraints for useful search strategies are discussed.
Measurement of amino acid composition of proteins by computerized microdensitometry of two‐dimensional electrophoresis gels
Authors: Latter Gerald I. , Metz Edward , Burbeck Stephen , Leavitt John 
Published: May 2005

AbstractA method which utilizes computerized microdensitometry to measure amino acid compositions of polypeptides resolved in two‐dimensional gels is described. Twenty cultures of two human cell lines were radiolabeled with each of the 20 different 14C or 35S – amino acids. Two‐dimensional polyacrylamide gel electrophoresis was performed on total protein samples of the cultures in replicates of three. For each amino acid label the relative amount of radioactivity in an “unknown” polypetide species was computed using the measured radioactivity simultaneously incorporated into the reference protein, β‐, γ‐actin, whose precise amino acid composition is known. We tested the accuracy of this method by computing the amino acid composition of two species of mutant β‐actin, which along with normal β‐, γ‐actin as the reference provided us with an ideal set of conditions. We found that amino acid composition can be predicted with a standard error of 6% over all twenty amino acids. Techniques are described to further reduce the measurement error so that compositions of proteins can be measured under less ideal conditions.
Direct digital imaging of radio‐labeled two‐dimensional gel beta emissions using micro‐channel plate image enhancement
Authors: Burbeck Stephen 
Published: May 2005

AbstractThe use of autoradiography to record the distribution of radio‐labels such as tritium or 35S in two‐dimensional (2‐D) gels has well known deficiencies: the film requires long exposures, has a nonlinear response curve, and has a restricted dynamic range. Autofluorography can improve upon each of these problems but only at the cost of resolution and quantitative accuracy. This paper demonstrates that a micro‐channel plate electron multiplier and a resistive anode position detector can detect and localize individual beta emissions from 35S or tritium with at least 200 μm resolution. The locations of these beta emissions can be collected, converted to a 2‐D image, and displayed on a graphics terminal or integrated over spot areas for quantitative estimates of spot intensity. The collection process is rapid, linear, and has a dynamic range far greater than that of film. With the 35S‐test gel (which required 40 h for an autoradiogram), strong spots can be integrated with better than ten percent accuracy with less than five minutes of data collection. Weak spots (those with less than 4 cpm) may require 80 min or more. Tritium gels require collection times an order of magnitude longer for equivalent quantitative accuracy.
Two‐dimensional polyacrylamide gel electrophoresis in micro‐gels: Gradient gel electrophoresis followed by dodecyl sulfate gel electrophoresis
Authors: Beltle Wolfgang , Zimmermann Sabine , Bopp Martin , Murach Karl‐Friedrich 
Published: May 2005

AbstractA miniature system for two‐dimensional polyacrylamide gel electrophoresis is presented. Discontinuous sodium dodecyl sulfate gel electrophoresis in micro‐slab gels following high resolution micro‐electrophoresis in cylindrical continuous polyacrylamide gradient gels separates macromolecules after an initial size fractionation into their subunits. The construction of a glass cuvette for micro‐slab gels and of a suitable electrophoresis tank is described in detail. Furthermore, procedures for silver staining and fluorography initially developed for gels of normal size were adapted to micro‐slab gels. Crude tissue extracts of Funaria hygrometrica L. Sibth. were analyzed without any further purfication. Twenty samples can be processed in about eight hours.
Isoelectric focusing in immobilized pH gradients for the determination of the genetic Pi (α1‐antitrypsin) variants
Authors: Görg Angelika , Postel Wilhelm , Weser Johann , Weidinger Sebastian , Patutschnick Walburgis , Cleve Hartwig 
Published: May 2005

AbstractThe genetic variants of the human protease inhibitor (Pi) system have been analyzed by isoelectricfocusing in immobilized pH gradients. With the use of a narrow pH range of pH 4.50 to 4.70 the six common PiM subtypes can be readily and reliably classified. The less common inherited Pi variants can be demonstrated most clearly in a gradient of pH 4.4 to 4.8. For the classification of PiZ a more basic pH gradient of 4.55 to 5.05 has been employed. Isoelectric focusing with immobilized pH gradients is also a useful tool for the study of the genetic variation in the Pi system. A number of problems to be adressed in future studies are discussed such as the delineation of further PiM subtypes, the detection of additional inherited heterogeneity, and the application of isoelectric focusing with immoblilized pH gradients in routine typing laboratories.
Selected buffer systems for moving boundary electrophoresis on gels at various pH values, presented in a simplified manner
Authors: Chrambach Andreas , Jovin Thomas M. 
Published: May 2005

AbstractMoving boundary electrophoresis on gels is useful for 4 purposes: (1) As an automatic concentration method to produce concentrated starting zones of proteins* for analytical and preparative resolving gels; (2) to provide a sharply defined reference zone in the resolving gel for Rf measurement; (3) for the preparative electrophoretic extraction of proteins from gel slices; (4) for high load preparative “charge fractionations” of relatively simple mixtures of proteins. It requires buffer systems at various pH values which are capable of setting up moving boundaries with numerically known leading and trailing ion mobilities. Moreover, since protein migration is slow compared with that of buffers, these moving boundaries have to have low displacement rates if they are to concentrate and align the proteins in order of mobility. Ten years ago, the authors made available in form first of magnetic tapes, then of microfiche, a collection of buffer systems for that purpose. But, in spite of the large number, over 4000, of these buffer systems, biochemists have not taken advantage of them widely. This appears in part due to the terminological complexity of the presentation needed to give an exhaustive physical‐chemical description of the systems. In part, it was due to the confusion about which of the various forms and techniques of moving boundary electrophoresis, known under a variety of names, these buffer systems applied to. Also, among the many buffer systems, only relatively few exhibited sufficiently low moving boundary displacement rates to allow slowly migrating proteins to migrate within the moving boundary. Finally, it proved difficult for potential users to interpret the magnetic tapes and microfiche; also tapes were excessively expensive. To remedy all these problems, and to allow biochemists to benefit more widely, both analytically and preparatively, from moving boundary electrophoresis at any pH, we have selected a small number of buffer systems capable of concentrating proteins within moving boundaries set up at various pH values, with low displacement rates, and with either anodic or cathodic displacement. These are presented in simplified format and terminology.
Direct isoelectric point determination of antigens by a new quantitative immunoelectrophoretic method
Authors: Guinet Ronald M. F. 
Published: May 2005

AbstractCrossed immunoelectrofocusing combines the high resolution of isoelectric focusing in the first dimension with the detection selectivity of quantitative immunoelectrophoresis through an antibody‐containing gel in the second dimension. A new quantitative immunoelectrophoretic method is described which permits in a single process simultaneous determination of isoelectric points and identification of individual proteins in a complex mixture. This method combines crossed‐line immunoelectrofocusing with crossed‐line immunoelectrophoresis on the same plate. Optimal conditions presented have been determined with our Candida tropicalis antigen‐antibody system.
Demonstration of activity for alkaline muscle ATPase in polyacrylamide gels using metal conversion methods
Authors: Kirkeby Svend , Moe Dennis 
Published: May 2005

AbstractATPase activity from skeletal muscle was tested at pH 9.2 in polyacrylamide gels using cobalt or lead conversion techniques. With cobalt it seems that the ion is precipitated both to acrylamide and to certain muscle components resulting in a reproducible but non‐enzymatic banding pattern. With the lead technique a specific strongly coloured band is present near the cathode. The band is suppressed by p‐hydroxymercuribenzoate but not by levamisole.
Two‐dimensional gel electrophoretic analysis of plasma proteins from a patient bitten by a rattlesnake
Authors: Bagley Elisabeth A. , Lonberg‐Holm Karl , Pandya Bharat V. , Budzynski Andrzej Z. 
Published: May 2005

AbstractWe have studied changes in plasma proteins in a patient bitten by a western diamondback rattlesnake (Crotalus atrox). Samples obtained at intervals during recovery were analyzed by “low resolution” two dimensional (2‐D) gel electrophoresis, a method which resolves at least 50 plasma components. The results show three days of complete afibrinogenemia, followed by gradual return of fibrinogen to a normal level. Both α‐ and β‐ lipoproteins also appeared altered or decreased in concentration during this period. An anomalous change in electrophoretic mobility of a portion of complement component 3 (C3) was detected during the first 15 days, but not at day 38. This modified protein was not C3b or C3c. A short incubation of human plasma or purified C3 with venom in vitro, did not mimic in vivo effects on either fibrinogen or C3, but plasma lipoproteins were modified directly in vitro. It is possible that the in vivo effects of venom on C3 and fibrinogen, as revealed by the 2‐D gel electrophoretic method, are indirect and require release or activation of cellular proteases.
Identification of mouse serum proteins increased by the administration of antitumor polysaccharide lentinan, by micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Takahashi Yuko , Okuyama Tsuneo , Maeda Yukiko Y. , Chihara Goro 
Published: May 2005

AbstractThe changes in mouse serum protein distribution after administration of lentinan, an antitumor polysaccharide, were examined using micro two‐dimensional electrophoresis in the absence of denaturing agents. Three protein spots were markedly increased in level and two of them were identified to be haptoglobin and haptoglobin partially bound with hemoglobin. Activity staining of haptoglobin after complex formation with hemoglobin and of ceruloplasmin were demonstrated.
Measurement of isoelectric point of S‐adenosyl‐L‐methionine and its metabolic products by an isoelectric focusing technique
Authors: Farooqui Jamal , Kim Sangduk , Paik Woon K. 
Published: May 2005

AbstractThe isoelectric point (pI) of S‐adenosyl‐L‐methionine and some of its metabolic products has been determined by an isoelectric focusing technique. The experimental pI values of S‐adenosyl‐L‐methionine and S‐methylmethionine are 7.24 and 6.85, respectively, and the pI values of S‐adenosyl‐L‐homocysteine and homocysteine are 6.05 and 5.65, respectively. This indicates that the contribution by adenosine, adenine and ribose to the total electric charge of S‐adenosyl‐L‐methionine or S‐adenosyl‐L‐homocysteine is approximately 0.40 pI unit. The differences in the pI values between two pairs of compounds (S‐adenosyl‐L‐methionine vs. S‐adenosyl‐L‐homocysteine and S‐methylmethionine vs. methionine) are very close (1.19 and 1.14, respectively). This difference is the amount of pI value contributed by the sulfonium group which has been formed by methyl addition. Surprisingly, the presence of a permanent positive charge at the sulfonium center in S‐adenosyl‐L‐methionine and S‐methylmethionine does not result in a higher pI values for these compounds. This might be due to the partial neutralization of the positive charge by the formation of carbanion. The mechanism of charge neutralization in these sulfonium compounds is discussed.
Studies of silver staining of human parotid saliva protein polyacrylamide gel electrophoretograms
Authors: Hallinan Frank M. 
Published: May 2005

AbstractThe silver staining procedure of Oakley et al. [3] has been applied to parotid saliva proteins and certain protein standards fractionated by isoelectric focusing and electrophoresis in denaturing and non‐denaturing polyacrylamide gels. This staining method was up to 100‐fold more sensitive than Coomassie Blue staining for sodium dodecyl sulphate‐containing gels and for focusing gels, after prefixation. In non‐denaturing gels, however, the relative sensitivity (i.e. silver vs. Coomassie) of different bands varies widely but this can be overcome by silver staining of gels after Coomassie Blue staining.
Agarose gel electrophoresis of bacteriophages and related particles. IV. An improved procedure for determining the size of spherical particles
Authors: Serwer Philip , Allen Jerry L. 
Published: May 2005

AbstractAttempts have been made to improve the accuracy with which sieving during electrophoresis in gels can be used to determine the size of viruses and related particles. Using as samples spherical bacteriophages and related particles of known radius (13.3–41.9 nm), it was found that electrophoretic mobility (μ) was a linear function of agarose concentration (A) in a hydroxyethylated agarose (SeaPlaque, Marine Colloids) for 0.4 ⩽ A ⩽ 1.6. The value of A extrapolated to a μ of 0 (A0) was a decreasing function of particle outer radius and was independent of the solid support‐free μ. Procedures for determining outer particle radius from values of A0 have been developed. When used with internal standards, these procedures measure outer particle radius with an accuracy of ±4 %.
Subtyping of human red cell phosphoglucomutase (PGM1) using conventional agarose gel electrophoresis
Authors: Bäur Walter , Dissing Jørgen 
Published: May 2005

AbstractA simple, rapid electrophoretic method for the subtyping of phosphoglucomutase (PGM1) isozymes is presented. Electrophoresis was performed in an agarose gel containing an acetic acid/L‐histidine/citric acid buffer system at pH 5.30 for 2.5 h. The separation of the isozymes was at least as good as that obtained by isoelectric focusing, but in contrast with that method, the electrophoretic procedure did not require expensive equipment or carrier ampholytes.
Two‐dimensional electrophoresis of serum proteins in myocardial infarction patients and subjects at risk of developing coronary heart disease
Authors: Gomo Zvenyika A. R. , Clark Penelope M. S. , Kricka Larry J. , Woods Kent , Buckley Brendan , Whitehead Thomas P. 
Published: May 2005

AbstractIso‐Dalt analysis of serum from myocardial infarction patients has revealed the presence of a group of low (<23 000) molecular weight basic peptides, as yet unidentified, which were not found in the serum of healthy controls. The presence and staining intensity of these proteins correlated with the qualitatively assessed serum creatine kinase MB isoenzyme activity. The proteins were not detected in sera from patients at risk of developing coronary heart disease (smokers, hypertensive patients, subjects with a family history of developing coronary heart disease, oral contraceptive users, hyperlipidaemics, and diabetics). This excludes their possible use as a predictive test for the development of coronary heart disease. It seems probable that they originate in the cells in or near the infarct.
The titration curve of native C reactive protein
Authors: Laurent Philippe , Potempa Lawrence A. , Gewurz Henry , Fiedel Barry A. , Allen Robert C. 
Published: May 2005

AbstractWe have studied the electrophoretic mobility of purified human C reactive protein (CRP) in a gradient from pH 3.0 to 9.0 produced by the titration curve method. The results suggest that the major species has a pI at pH 6.3 ± 0.2. From pH 7.2 to 8.5, CRP was at its isoelectric point and did not migrate from the application trough. From pH 4.0 to 5.0, CRP produced a hazy, diffuse pattern most probably due to instability in the region close to the isoelectric point of the acid‐denatured form of the molecule. Below pH 4 CRP resolubilized to assume heterogenic anionic forms.
Separation of legumin and vicilin, the storage proteins from germinating Vicia faba seeds, by electrophoresis on cellulose acetate membranes
Authors: Leslie Philip , Bonner Richard , Vandevalle Ian , Olsson Richard 
Published: May 2005

AbstractConventional electrophoretic methods of assessing the purity of a protein are both expensive and laborious. The method of zonal isoelectric precipitation electrophoresis involves precipitation of the protein under investigation at its isoelectric point after application onto cellulose acetate membranes. Contaminating proteins will be mobile and are easily visualized by conventional staining methods. Application of zonal isoelectric precipitation electrophoresis to the storage proteins of Vicia faba seeds (legumin and vicilin) has proved valuable in this laboratory, and, by adjustment of the buffering system, can be made to work for other protein mixtures.
The effects of freezing and thawing on lactate dehydrogenase and esterase isozyme patterns of the goldfish Carassius auratus, demonstrated by isoelectric focusing
Authors: Burch Simon J. , Lawson Ronald , Davies David H. 
Published: May 2005

AbstractCurrent literature suggests that the reliability of results obtained from electrophoretic studies using material which has been frozen is in doubt. Banding patterns of lactate dehydrogenase and non‐specific esterase produced by isoelectric focusing (IEF) of fresh tissues from the goldfish, Carassius auratus, are presented. Changes which occur in these patterns due to repeated freezing and thawing involve a reduction in both the intensity and the number of bands. Differences in the IEF profiles are extensive and consistent. Changes also occur in material which has been frozen for a ten‐week period and thawed once. Storage in liquid nitrogen, ‐180°C, as distinct from a conventional freezer at ‐20°C, produes no significant improvement. Interpretation of results from material that has been frozen must therefore be undertaken with extreme care, especially when sensitive separation techniques like IEF are used.
A rapid detection system for the quantitative evaluation of radioactively labeled proteins in dehydrated slab gels
Authors: Kruppa Joachim 
Published: May 2005

Abstract3H‐ and 35S‐Labeled proteins of vesicular stomatitis virus were separated by polyacrylamide gel electrophoresis, localized and directly quantified by the Linear Analyzer after dehydration of the gel. The counting efficiency of the Linear Analyzer was approximately 0.1% for tritium in polyacrylamide gels, and increased after blotting to 2‐3%, whereas the detection efficiency for 35S was 2‐3% in polyacrylamide gels. An increase in gel thickness reduced the detection efficiency of the Linear Analyzer significantly, an effect which was also observed in fluorography. The instrument can be used for direct, quantitative evaluation of tritium in gels and blots because a linear relationship exists between the input radioactivity and the recorded counts. Double‐label experiments with 3H‐ and 35S‐labeled proteins can also be quantified. The spatial resolution of the Linear Analyzer reaches the quality attained in fluorography. Quantitative evaluation of gels and blots by the Linear Analyzer has the additional advantage of being less time‐consuming than detection by fluorography.
Analysis of human haptoglobin‐hemoglobin complexes by micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Takahasi Yuko , Okuyama Tsuneo 
Published: May 2005

AbstractA microscale‐multisample two‐dimensional electrophoretic technique, in which no denaturing agent is used, was applied to the analysis of three phenotypes of haptoglobin and their hemoglobin complexes in human sera. Haptoglobin‐hemoglobin complexes did not dissociate during the electrophoretic run, so their pIs and apparent molecular weights could be estimated. The number of spots of haptoglobin‐hemoglobin complex derived from a haptoglobin molecular species coincided with the number of β chains involved in it. Heterogeneity in pI of the haptoglobin‐hemoglobin complexes was observed, which suggests the heterogeneous binding of an αβ half‐molecule of hemoglobin to a β chain of haptoglobin.
Improved protein analysis on nitrocellulose membranes
Authors: Polvino William J. , Saravis Calvin A. , Sampson Christian E. , Cook Richard B. 
Published: May 2005

AbstractThe use of peroxidase‐conjugated antibodies for detecting specific tumor associated antigens bound to nitrocellulose membranes is being increasingly used in our laboratory. This method offers many advantages. However, we have observed several problems which can cause confusing results and possible misinterpretations. Some methods of eliminating artifacts are described as well as the use of glutaraldehyde vapor fixation as a means to increase sensitivity.