Discontinuous Buffer System Research Articles

Resolution of Bacterial Proteins by Polyacrylamide Gel Electrophoresis on Slabs: MEMBRANE, SOLUBLE, AND PERIPLASMIC FRACTIONS

The application of a method of electrophoretic protein analysis to the investigation of the total protein composition of Salmonella typhimurium is described. It involves electrophoresis of many samples simultaneously on a thin slab of acrylamide gel, in the presence of sodium dodecyl sulfate, with the discontinuous buffer system of Laemmli ((1970) Nature 227, 680–685). Up to about 25 samples can be run simultaneously, thus improving both efficiency and ability to compare patterns. The sensitivity of the method for detection of changes both in soluble proteins and the insoluble membrane proteins of the cell is shown. Membrane preparations from the wild type strain and many different mutant strains have been analyzed. The major membrane proteins observed and their relationship to proteins described in the literature are discussed. The marked changes detected in a variety of mutant strains and under a variety of growth conditions are also discussed, either as to their specific meaning or as evidence of the effectiveness of the method. Solubilization artifacts concerning the proteins of the outer cell membrane are presented. The membrane fractions from two Escherichia coli strains have also been analyzed.

Detection and identification of diagnostic Histoplasma capsulatum precipitates by counterelectrophoresis.

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.

Multiple forms of acetylcholinesterase from pig brain.

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.

Multiphasic zone electrophoresis, IV. Design and analysis of discontinuous buffer systems with a digital computer.

Annals of the New York Academy of SciencesVolume 209, Issue 1 p. 477-496 MULTIPHASIC ZONE ELECTROPHORESIS. IV. DESIGN AND ANALYSIS OF DISCONTINUOUS BUFFER SYSTEMS WITH A DIGITAL COMPUTER Thomas M. Jovin, Thomas M. Jovin Abteilung für. Molekulare Biologic Max-Planck-lnstitut für Biophysikalische Chemie 34 Göttingen, Federal Republic of Germany This work was done during the tenure of an Established Investigatorship of the American Heart Association. The Appendix was written by George Striker.Search for more papers by this author Thomas M. Jovin, Thomas M. Jovin Abteilung für. Molekulare Biologic Max-Planck-lnstitut für Biophysikalische Chemie 34 Göttingen, Federal Republic of Germany This work was done during the tenure of an Established Investigatorship of the American Heart Association. The Appendix was written by George Striker.Search for more papers by this author First published: June 1973 https://doi.org/10.1111/j.1749-6632.1973.tb47551.xCitations: 104 This work was done during the tenure of an Established Investigatorship of the American Heart Association. The Appendix was written by George Striker. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume209, Issue1Isoelectric Focusing and IsotachophoresisJune 1973Pages 477-496 RelatedInformation

Multiphasic zone electrophoresis. 3. Further analysis and new forms of discontinuous buffer systems.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMultiphasic zone electrophoresis. III. Further analysis and new forms of discontinuous buffer systemsThomas M. JovinCite this: Biochemistry 1973, 12, 5, 890–898Publication Date (Print):February 27, 1973Publication History Published online1 May 2002Published inissue 27 February 1973https://doi.org/10.1021/bi00729a016RIGHTS & PERMISSIONSArticle Views170Altmetric-Citations61LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (996 KB) Get e-Alerts Get e-Alerts

Multiphasic zone electrophoresis. II. Design of integrated discontinuous buffer systems for analytical and preparative fractionation.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMultiphasic zone electrophoresis. II. Design of integrated discontinuous buffer systems for analytical and preparative fractionationThomas M. JovinCite this: Biochemistry 1973, 12, 5, 879–890Publication Date (Print):February 27, 1973Publication History Published online1 May 2002Published inissue 27 February 1973https://doi.org/10.1021/bi00729a015RIGHTS & PERMISSIONSArticle Views183Altmetric-Citations67LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Detection and Identification of Diagnostic Histoplasma capsulatum Precipitates by Counterelectrophoresis

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.

Experimental Validation of the Predicted Properties of a Multiphasic Buffer System Applied to Polyacrylamide Gel Electrophoresis

Abstract The properties of a buffer system predicted by T. M. Jovin's theory of multiphasic (discontinuous) buffer systems have been tested experimentally both in free solution and in polyacrylamide gels. The most widely applied multiphasic buffer system, viz., the Tris-glycine system of Ornstein and Davis was used for this purpose. The properties of this system were computed on the basis of the Jovin theory. The pH, specific conductance, and boundary displacement were measured in the buffers corresponding to the original and the operative buffers of both the stacking (upper) gel and the separation (lower) gel in polyacrylamide gel electrophoresis. Values of pH and specific conductance in the absence of gels were found to be in reasonable agreement with theory. In polyacrylamide gels, after removal of residual reactants of the polymerization reaction by equilibration with the appropriate buffer, there was good agreement between observed and predicted values of pH and boundary displacement. However, the ob...

Cellular and secretory proteins of the salivary glands of Sciara coprophila during the larval-pupal transformation.

The cellular and secretory proteins of the salivary gland of Sciara coprophila during the stages of the larval-pupal transformation were examined by electrophoresis in 0.6 mm sheets of polyacrylamide gel with both SDS-continuous and discontinuous buffer systems. After SDS-electrophoresis, all electrophoretograms of both reduced and nonreduced proteins from single glands stained with Coomassie brilliant blue revealed a pattern containing the same 25 bands during the stages of the larval-pupal transformation. With the staining procedures used in this study, qualitative increases and decreases were detected in existing proteins and enzymes. There was no evidence, however, for the appearance of new protein species that could be correlated with the onset of either pupation or gland histolysis. Electrophoretograms of reduced samples of anterior versus posterior gland parts indicated that no protein in the basic pattern of 25 bands was unique to either the anterior or posterior gland part. Electrophoretograms of reduced samples of secretion collected from either actively feeding or "cocoon"-building animals showed an electrophoretic pattern containing up to six of the 25 protein fractions detected in salivary gland samples, with varied amounts of these same six proteins in electrophoretograms of secretion samples from a given stage. Zymograms of non-specific esterases in salivary gland samples revealed a progressive increase in the amount of esterase reaction produce in one major band and some decrease in the second major band during later stages of the larval-pupal transformation.

Detection of fibrinogen-fibrin degradation products by counterelectrophoresis.

Counterelectrophoresis using a discontinuous buffer system permits detection of fibrinogen-fibrin degradation products (FDP) under a variety of clinical circumstances. The method is sensitive, reliable, and is easily performed using conventional equipment in any clinical laboratory assuming the responsibility for assaying fibrinogen-fibrin degradation products.

The non-histone proteins of chromatin: Their isolation and composition in a number of tissues

1. 1. A method is described for the fractionation of salt-urea-dissociated chromatin using hydroxylapatite. With the exception of experiments using chromatins prepared from “citric acid” nuclei, high yields of acidic non-histone proteins, relatively free of RNA, can be obtained by this procedure. 2. 2. The non-histone proteins of a number of chromatins were compared by electrophoresis in sodium dodecyl sulphate-urea polyacrylamide gels employing a discontinuous buffer system. Proteins from mouse chromatins prepared from “citric acid” nuclei were found to be extremely heterogeneous, but in the case of calf thymus the proteins were mainly low molecular weight. On the other hand, the non-histone proteins of chromatin from “sucrose” nuclei appeared to contain fewer high molecular weight species in the tissues studied, with the exception of brain. Preparation of nuclei by the double-detergent procedure of Penman ( J. Mol. Biol., 17 (1966) 117) gave chromatin with a low protein to DNA ratio. These proteins also appeared to be predominantly low molecular weight. Duck erythrocyte nuclei prepared by lysis also contained low molecular weight chromatin non-histone proteins. 3. 3. Using salt fractionation techniques attempts were also made to remove “cytoplasmic” and “residual” acidic proteins from chromatin. The proteins which remained with the DNA and histones were found to be mainly low molecular weight in kidney and liver, but in the case of brain a wide spectrum of proteins was seen. 4. 4. Little tissue or species specificity of non-histone proteins were found on comparison of “sucrose” nuclei chromatins prepared from a number of mouse and bovine tissues. 5. 5. It is concluded that the non-histone proteins which remain tightly bound to DNA in chromatin are of the same approximate size as the basic histones. Because of the procedural variation in the heterogeneity of these chromatin proteins, detection of single species of regulatory proteins appears to require other techniques.

Vertical Plate Gel Electrophoresis for the Differentiation of Meat Species

Abstract Polyacrylamide gel electrophoresis of fresh meat sarcoplasmic proteins by the vertical plat e technique was studied as a method for the detection and identification of pure and mixed species extracts. A single 7% Cyanogum gel containing 1M urea, in conjunction with a tris-chloride, tris-glycine discontinuous buffer system, produced satisfactory resolution of single species and mixed species muscle protein extracts. Eleetrophoretie patterns were obtained by direct analysis of meat proteins extracted with a tris-chloride-10% glucose extraction buffer at pH 6.7. Mixtures of beef and pork and beef and horse were easily identified. The possibility for a d a p t i n g t h e eleetrophoretie procedure to the analysis of cooked meat products and non-meat proteins is discussed.

Vertical Plate Gel Electrophoresis for the Differentiation of Fish and Shellfish Species

Abstract The vertical plate polyacrylamide gel electrophoresis technique described for differentiation of meat was applied to fresh and frozen fish and shellfish species. A single 7% Cyanogum gel containing mercaptoethanol, in conjunction with a tris-chloride, tris-glycine discontinuous buffer system, produced satisfactory resolution of fish and shellfish species extracts. The electrophoretic patterns were obtained by direct analysis of the proteins extracted with a tris-chloride-10% glucose extraction buffer at pH 6.7. This technique can also be used to detect substitution of one fish or shellfish species for another.

Rapid Large-Scale Screening of Blood Proteins by Use of Cellulose Acetate Electrophoresis on a Solid Support

Abstract We describe a new principle of cellulose acetate electrophoresis (solid-support electrophoresis), in which cellulose acetate membranes are given a solid support, instead of being suspended as heretofore. The new technique is particularly well suited for screening procedures, because many samples can be processed economically on a single run. Also described for the first time, in conjunction with solid-support electrophoresis, is a method of (a) simultaneously using continuous and discontinuous buffer systems with specially (series) oriented membranes, or (b) a single-sample application for fractionating different constituents simultaneously (proteins, lipoproteins, isoenzymes, etc.).

Discontinuous buffer systems for analytical and preparative electrophoresis of enzymes on polyacrylamide gel

Discontinuous buffer systems for analytical and preparative electrophoresis of enzymes on polyacrylamide gel

Resolution of prorennin and rennin by polyacrylamide gel electrophoresis

The heterogenity of prorennin and rennin was investigated with polyacrylamide gel electrophoresis. A discontinuous imidazole buffer system at pH 7.1 was developed which, in conjunction with efficient refrigeration of the gel, was compatible with the stability of prorennin and rennin. This technique separated rennin and prorennin and demonstrated that both the enzyme and zymogen were heterogeneous. Rennin was resolved into four components in 6.5% polyacrylamide gels, while prorennin was shown to consist of three electrophoretically distinct bands. Electrophoretic analysis of the autocatalytic activation of prorennin at pH 5.0 demonstrated the conversion to rennin and indicated that each zymogen component gave rise to a separate rennin.

IMMUNOELECTROOSMOPHORESIS USING DISCONTINUOUS BUFFER SYSTEM TO DETECT AUSTRALIA ANTIGEN IN POOLED HUMAN PLASMA

IMMUNOELECTROOSMOPHORESIS USING DISCONTINUOUS BUFFER SYSTEM TO DETECT AUSTRALIA ANTIGEN IN POOLED HUMAN PLASMA

Separation of purified human chorionic gonadotropin into single bands by isoelectric focusing and their characterization

During the past two years several investigators have reported further success in purification and characterization of human chorionic gonadotropin (HCG) [l-3]. Recently we described a chromatographic method for the purification of HCG [4], which is similar to the procedure described by Van Hell [5] . We found however, that the highly purified hormone is still inhomogeneous in starch gel electrophoresis using discontinuous buffer systems and in disc electrophoresis in presence of 6 M urea. Furthermore, subfractions of native [6] and modified [7] HCG have been isolated after treatment with 8 M urea. This paper reports the separation of purified HCG into 6 single bands by preparative isoelectric focusing with thin layer electrophoresis in Sephadex. Up to now the bands have been characterized by their IP, content of N-acetyl neuraminic acid (NANA) and biological activity. Moreover the results obtained by refocusing and disc electrophoresis provides additional information about the subunit pattern of HCG.

Molecular Weight Determination of Protein-Dodecyl Sulfate Complexes by Gel Electrophoresis in a Discontinuous Buffer System

This report describes methods and results obtained by combining the techniques of sodium dodecyl sulfate (SDS) gel electrophoresis and electrophoresis in discontinuous buffer systems. The SDS gel system utilizes a sulfate-borate discontinuity which stacks and unstacks protein-SDS complexes over a range of 2,300 to 320,000 daltons, providing high resolution fractionation. The properties of protein-SDS complexes are investigated by calculating retardation coefficients and apparent free mobilities from Ferguson plots. Apparent free mobilities are approximately constant, establishing a linear relationship between the logarithm of the relative mobility and the retardation coefficient. The retardation coefficient is shown both empirically and theoretically to be a uniform function of molecular weight of protein-SDS complexes over specified ranges, providing a rationale for determining molecular weight from plots of the negative logarithm of relative mobility against molecular weight.

Synaptosomal proteins and axoplasmic flow: Fractionation by SDS polyacrylamide gel electrophoresis

The neuronal cell body is known to supply the axon and the nerve terminal with various materials which migrate through the axon at several different velocities. A perikaryal origin of an important part of the syn- aptic proteins has been suggested and substantiated [1 ]. Using the visual system of the pigeon as a model, it was shown that proteins labelled in one retina reach the optic nerve terminals in the contralateral optic lobe in at least two waves. The first, corresponding to the fast axoplasmic flow, has a peak at 24 hr while the second, having the characteristics of slow axo- plasmic flow, has its maximum at 14 days [2-4], Previous attempts to fractionate the synaptosomal proteins [5-8] have not dealt with the source of origin of these proteins, i.e. the problem was not in- vestigated whether the proteins arrive with the fast or slow axonal flows or whether they are synthesized in the nerve endings. This report is concerned with the characterization, by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, of the synaptic proteins migrating from the perikaryon. 2. Materials and methods Each pigeon was injected intraocularly [2] with 50/aCi of aH-leucine (1-4,5-3H-leucine, specific activity 22 Ci[mM) or 50/aCi 14C-leucine (1-1- 14C- leucine, specific activity 62 mCi/rnM, both Radio- chemical Centre, Amersham, England) in 50 pl H20. Subcellular fractionation of the optic lobe's homo- genate was performed by the slightly modified [2] method of Gray and Whittaker [9]. The fraction enriched in synaptosomes, containing about 1.5 mg protein, was solubilized in 200/al of SUCK solution (1% SDS, 8 M urea, 0.01 M dithiotreitol and 0.05 M K2CO3) [10~ 11] by gentle agitation at room temper- ature for 30 min and used immediately for the electro- phoresis. An ORTEC SLAB electrophoresis unit was used with a pulsed constant-power source. 0.1% SDS acrylamide discontinuous gradient gel was prepared according to Grossfeld and Shooter [12]. In each cell, 7.5 ml of 12% acrylamide, 7.5 ml of 9.6%, 6 ml of 7.5% and 3 ml of 4.2% were layered on top of each other and polymerized simultaneously. The stacking gel, 8 ml of 2.5% acrylamide, was then polymerized with light. 50/al of synaptosomes solubilized in SUCK were applied on the stacking gel. Electrophoresis was performed in the discontinuous Tris glycine buffer system [12] using 300 pulses/see at 120 V, 10 mA and 0.1 /aF discharge capacitance. The proteins moved towards the anode at room temperature for 16 hr. The gels were fixed in concentrated acetic acid for 12 hr and stained with 1% amido black B in 7% (v/v) acetic acid for 3 hr. They were destained in 7% acetic acid and sectioned between successive bands, where mechanical separation was possible. The pieces of gel were solubilized [13] and counted in a Mark I Nuclear Chicago Scintillation Counter. The dpm were