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PubMed1976

The demonstration of asymmetric hemoglobin hybrids by polyacrylamide electrophoresis.

A simpler, more economical technique than previously reported, that of conventional polyacrylamide gel electrophoresis alone, is described for the detection of asymmetric hemoglobin hybrids of the forms alphaXalphaYbeta2 and alpha2betaXbetaY when bloods from individuals with alpha and beta chain variants were examined. The presence of alpha chain variant hybrids, never before reported, is further evidence that hybrid formation is a more widespread phenomenon than has previously been thought of. Hybrids were found in artificial mixtures of hemoglobins and more importantly, are also reported here for the first time in bloods of individuals heterozygous for hemoglobin variants. These hybrid tetramers were as stable as the parent hemoglobins when examined under anaerobic conditions. The involvement of HbF in the formation of hybrids of the type alpha2betagamma is reported, and an analysis of the possible role of these as well as alpha2betaAbetaS hybrids in the sickling process is presented.

Binding Sites↗
PubMed1976

Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel.

Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.

Amino Acids↗
PubMed1977

Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Standard experimental procedures for continuous polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were modified to give more effective separation and improved resolution of myofibrillar proteins. The system utilizes a running gel consisting of 10% acrylamide with 0.1% bisacrylamide crosslinker (100:1) incorporating 400 mM Tris/glycine (pH 8.80), 0.1 mM ethylenediaminetetraacetate, 5% glycerol and 0.1% sodium dodecyl sulfate. Electrophoresis was performed at 1 mA per gel with corresponding running times of 4-6 h. The myosin heavy chain enters and migrates as a narrow symmetrical band while the smaller regulatory proteins of the myofibril are resolved. The utility of the procedure in relation to the study of protein structure is detailed.

Animals↗
PubMed1977

Immunological identification of complex proteins resolved by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis.

Immunological identification of an antigen resolved from a protein complex by sodium dodecyl sulfate polyacrylamide gel electrophoresis has been attained. The identification is based on the formation of immunoprecipitin lines after the antigen diffuses laterally from acrylamide gel transverse slices into a surrounding agarose gel. This technique was designed for study of contractile and regulatory protein complexes of non-muscle cells where the scarcity of tissue precludes easy purification or high yield of muscle-like proteins. It complements double-gel immunodiffusion or immunoelectrophoresis and its use may be extended to other protein complexes.

Actinin↗
PubMed1977

Quantitative polyacrylamide gel electrophoresis and specific activities of human somatotropin and its derivatives.

A preparation of human pituitary somatotropin examined in quantitative polyacrylamide gel electrophoresis is conformationally compact. The derived molecular weight by quantitative electrophoresis is consistant with the known mass of the hormone and value obtained by other methods. A plasmin-modified somatotropin is more compact and shows full activity in immunoassay, and in lymphocyte binding and somatotropic assays, with enhanced lactogenic activity. The N-terminal 134-amino acid fragment and a hendekakaihekaton fragment exist in non-monomeric forms. The N-terminal fragment has immunologic and biologic activity, with greatest activity in the in vivo somatotropic assay. The hendekakaihekaton fragment exhibited only marginal activity. All preparations showed heterogeneity of charge in quantitative electrophoresis with discreet charge isomerism recognizable for the native and plasmin-modified preparations.

Binding Sites, Antibody↗
PubMed1978

Multiple isoferritins in mouse liver: demonstration by polyacrylamide gel electrophoresis.

Unfractionated pure ferritin isolated from the livers of female or male mice forms five narrow protein-positive bands on standard 5% polyacrylamide gel disc electrophoresis (pH 9.0). Since all of these sub-bands also contain iron, they are interpreted as being isoferritins. The multiple sub-bands are very unlikely to be artifactually generated by the analytical procedure used since they are not found in horse spleen ferritin when this is coelectrophoresed with female mouse liver ferritin. The present results provide an independnet indication that many isoferritins indeed can be found within a single organ.

Animals↗
PubMed1979

Resolution of the glycosyltransferase activities from two strains of Streptococcus mutans by polyacrylamide gel electrophoresis in the presence of Tween 80.

The glycosyltransferase complex from Streptococcus mutans can be resolved by polyacrylamide gel electrophoresis following treatment with Tween 80. The enzyme complex was treated with Tween 80 and electrophoresis was performed in the presence of Tween 80. Discrete bands of glycosyltransferase activity were observed when the gels were incubated in buffered sucrose. The enzyme aggregation that occurs with the exocellular glycosyltransferase may involve lipophilic interactions that are disrupted by the Tween 80.

Electrophoresis, Polyacrylamide Gel↗
PubMed1979

Studies of dynein from Tetrahymena cilia using agarose polyacrylamide gel electrophoresis.

Described in this report is an application of agarose-polyacrylamide gel electrophoresis, which separates protein components of crude dynein fraction (Fraction I by Gibbons) derived from Tetrahymena cilia. By this method, the fraction was separated into three protein components (designated as bands I, II and III) on the gel. When the gel was actively stained for dynein ATPase, a single band appeared, which coincided with the position of band I. A purified dynein prepared by controlled pore glass (CPG-10) column chromatography and followed by Biogel A-15m filtration showed one band on the gel at the same position as band I. These results suggest that among these three protein components, band I represents dynein and bands II and III are derived form non-ATPase protein. 'Burstic phenomenon' was also observed on their ATPase activity when axoneme or crude dynein fractions were used for ATPase assay, while the phenomenon was almost extinguished when partially purified dynein after controlled pore glass column chromatography was used as sample.

Adenosine Triphosphatases↗
PubMed1979

Factors affecting the electrophoretic mobility of the major outer membrane proteins of Escherichia coli in polyacrylamide gels.

The outer membrane proteins of Escherichia coli can be resolved by polyacrylamide gel electrophoresis in the presence of anionic detergents. Factors such as the choice of detergent and buffer system and the presence of urea in the separation gel are all shown to affect the charge and/or the configuration of the detergent-protein complexes and will affect the relative migration of these complexes to different extents. The procedures described in this paper may be of use in the determination of the relatedness of the proteins from the same or different strains. In addition, detailed examinations of the effects of these different parameters and the effect of changes in acrylamide concentrations may be useful in the detection of unusual characteristics which may indicate the presence of posttranslational modification.

Cell Membrane↗
PubMed1979

Microheterogeneity of rat, mouse and human alpha1-fetoprotein as revealed by polyacrylamide gel electrophoresis and by crossed immuno-affino-electrophoresis with different lectins.

Polyacrylamide gel electrophoresis and crossed immuno-affino-electrophoresis with several free lectins have been used to characterize and to compare the molecular heterogeneity of rat, mouse and human alpha1-fetoproteins. Each alpha1-fetoprotein contains a variable number of electrophoretic variants depending on the gel porosity. In SDS electrophoresis, two molecular size populations are present in rat alpha1-fetoprotein (Mr = 74 000 and 72 000) and in mouse alpha1-fetoprotein (Mr = 73 000 and 72 000) but only one is observed in human alpha1-fetoprotein (Mr = 70 000). The crossed immuno-affino-electrophoresis patterns square with affinity chromatography results and reveal a marked and characteristic heterogeneity for the three alpha1-fetoprotein species with Concanavalin A, Ricinus communis and Lens culinaris lectins. No lectin-alpha-fetoprotein interaction is apparent with Ulex, Lotus and wheat germ lectins. Since similar patterns are obtained whether with purified alpha1-fetoprotein or with unfractionated fresh fetal sera, it is likely that this heterogeneity is not a consequence of artefactual molecular modifications arising during the purification procedure.

Animals↗
PubMed1980

A specific stain for sulfhydryl groups in proteins after separation by polyacrylamide gel electrophoresis.

A new method is described for specifically staining protein sulfhydryl groups after the proteins have been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in slab gels. The stain will detect as little as 0.25 microgram of lysozyme and 1 microgram of most other proteins; the range of sensitivity for a specific protein depending on its sulfhydryl content. Proteins with no cysteine residues (type I collagen) and glycoproteins do not cause spurious staining.

Cysteine↗
PubMed1980

Solubilization of oil-soluble dyes by sodium dodecyl sulfate-protein polypeptide complexes with reference to SDS-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate binds to the linear polypeptide derived from a protein to form micelle-like clusters (Takagi, T., Tsujii, K. and Shirahama, K. (1975) J. Biochem. 78, 939--947) which are expected to solubilize lipophilic materials. Such clusters were found to afford the complexes strong solubilizing power against oil-soluble dyes, comparable or slightly superior to SDS micelles, by conventional solubilizing technique (equilibration with solid dye) and by gel chromatography technique first applied to measure solubilization by such complexes. Solubilization behavior of the bound SDS was insensitive to the kind of protein polypeptide, in contrast to the variety found in the pioneering similar study on the complexes between SDS and initially native proteins (Steinhardt, J., Stocker, N., Carroll, D. and Birdi, K.S. (1977) Biochemistry 16, 718--725). The knowledge of the solubilizing power of SDS-protein polypeptide complexes seems to be valuable in design of experiments and interpretation of results obtained in application of SDS-polyacrylamide gel electrophoresis to samples containing lipophilic materials.

Colloids↗
PubMed1981

Separation and localization on sodium dodecyl sulfate polyacrylamide gels of pig spleen lymphocyte plasma membrane proteins which bind 125I-labelled phytohemagglutinin.

The protein constituents of splenic pig lymphocytes derived from the plasma membranes, the endoplasmic reticulum or from a non-ionic (Nonidet P-40) detergent of whole cells have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and non-reducing conditions. The binding of Phaseolus vulgaris (red kidney bean) phytohemagglutinin to these constituents has been studied by incubation of the labelled lectin with the undried electrophoretograms. Results show characteristic phytohemagglutinin-reactive components in each cell fraction, although the majority of the lectin-binding bands are common to the three fractions. Comparison of binding patterns for the reduced and non-reduced electrophoretograms does not reveal significant differences between the binding profiles. The components which bind the lectin in the largest amounts are located in the upper halves of the gels, which correspond to a molecular range of (75-250) . 10(3) daltons. Our data suggest that pig spleen lymphocyte plasma membranes contain at least 25-30 glycoproteins which can bind phytohemagglutinin. Iodine-labelled phytohemagglutinin binds to the vesicles prepared from lymphocyte plasma membranes and the Scatchard plot shows a non-linear upward concave curve.

Animals↗
PubMed1983

Effect of dithiothreitol on the 1 alpha, 25-dihydroxyvitamin D3 chick intestinal receptor analyzed by polyacrylamide gel electrophoresis.

This is the first report of the use of non-denaturing polyacrylamide gel electrophoresis (PAGE) to measure the apparent molecular weight of the chick intestinal 1 alpha, 25-dihydroxyvitamin D3 (1, 25-(OH)2 - D3) receptor and to study the effect of dithiothreitol on it. When prepared in the absence of this factor, chick intestinal cytosol contained one major specific 1,25 - (OH)2 - D3 binding peak. Its apparent molecular weight was 95,200 +/- 1,900 (SD) daltons. Preparation of the cytosol in the presence of 5 mM dithiothreitol resulted in the appearance, besides the 95,000 daltons peak, of an additional 1,25 - (OH)2 - D3 binding peak, the molecular weight of which was 73,600 +/- 3,300 (SD). This effect of dithiothreitol could be suppressed by the simultaneous addition of 10 mM N alpha-p-tosyl-L-arginine methyl ester (TAME), a protease inhibitor.

Animals↗
PubMed1985

Monoclonal antibodies, electrophoretically transferred from polyacrylamide gels, retain their ability to bind specific antigens.

Antibodies subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper, have been found to retain the ability to bind specific antigen. This has been demonstrated for two groups of antibodies, directed to a) a large molecular weight glycoprotein of the human milk fat globule and b) human interferon alpha 2. Immunoreactive antibody fragments produced by protease digestion could also be identified in this way on Western blots, thus permitting the development of optimal conditions for digestion, without the need for extensive purification procedures.

Antibodies, Monoclonal↗
PubMed1991

Electrophoretic separation of S. pombe chromosomes in polyacrylamide solutions using a constant field.

Previous electrophoretic separations of megabase (Mb) sized DNA have been achieved in pulsed electric fields, using agarose gel as a matrix. The present study demonstrates separations of Mb sized DNA due to a retardation of migration in proportion to the concentration of uncrosslinked polyacrylamide of 5 x 10(6) molecular weight, using a constant electric field. Potentially, the method should be applicable to large DNA in general, greatly reducing the instrumental complexity of such separations and rendering them compatible with capillary electrophoresis apparatus.

Chromosomes, Fungal↗
PubMed1985

Identification of denatured enzyme proteins in sodium dodecyl sulfate polyacrylamide gels.

A simple modification of the immunological sandwich method of Muilerman et al. for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal alpha-glucosidase as a model system are described. The method was applied to identify a protein of Mr 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.

Electrophoresis, Polyacrylamide Gel↗