Protein separated by polyacrylamide gel

Silver-Stain Detection of Proteins Separated by Polyacrylamide Gel Electrophoresis... 

Silver-stained polyacrylamide gels " " have been used in quantitative and fast separations of individual proteins labelled with weak -emitters H) by polyacrylamide gel electrophoresis. The amount of radioactivity in a given band can be determined rapidly by liquid scintillation counting, which was found to be independent of the silver deposition and total protein content, but hnear with respect to the amount of radioactivity in the given band, thus avoiding the lengthy exposures needed, for instance, in the autoradiography of [ H]methionine-labelled proteins separated by polyacrylamide gel electrophoresis. The method was used to determine [ H]mannose and [ H]fucose incorporated into a cell adhesion protein . 

Fig. 2. Alternation ot gel-shitt and filter-binding selection steps Target-bound and unbound radiolabeled RNA aptamers are separated by polyacrylamide gel electrophoresis, visualized by autoradiography, purified from the gel, and used for the subsequent nitrocellulose-filter binding selection step. The experiments are earned out in the presence (-i-) and absence (-) of target protein using the SELEX cycles 0 (control), 3, and 7. The figure illustrates the increase of binding affinity of selected RNA pools, seen as augmented quantity of RNA retained together with the receptor protein at the top of the gel (modified from ref. (8)).

Transfer of proteins onto nitrocellulose or PVDF membranes is usually performed after separation of complex protein mixtures by polyacrylamide gel electrophoresis (PAGE). Proteins can be separated on the basis of their molecular weight or isoelectric point, under reducing or nonreducing conditions, and it is therefore for the investigator to establish the most appropriate separation conditions for particular samples. Full details of PAGE can be found in refs. 4 and 5, and the reader is encouraged to use these as sources of further details... 

MAPK is active only when phosphorylated on two specific amino acids, one threonine and one tyrosine. Because of this, MAPK activity can be measured by determining the content of phosphotyrosine in the enzyme using highly specific antiphosphotyrosine antibodies. Known amounts of protein are separated by polyacrylamide gel electrophoresis, transferred to sheets of nitrocellulose (or polyvinylidine difluoride), and probed with antiphosphotyrosine antibodies. To increase the signal produced by the detection system, it may be necessary to immunoprecipitate MAPK from known quantities of tissue, or to partially purify MAPK, biochemically, before immunoblot analysis. An increase in specific antiphosphotyrosine antibody binding implies that MAPK activity increases. 

The bulk proteins of guinea pig seminal vesicle secretion that are soluble in 150 mM NaCl have been separated by ion-exchange chromatography and other techniques including gel or paper electrophoresis under nondenaturing or denaturing conditions (7, 160-166). In sharp contrast to rat vesicular secretion proteins (see below), prior exposure of whole or saline-soluble guinea pig seminal vesicle secretion proteins to thiols does not alter the profiles of bands separable by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate with or without concentrated urea (J. Wilson and H. G. Williams-... 

Antigens are separated by polyacrylamide gel electrophoresis, electroblotted onto a membrane and then incubated with serum samples from subjects. Antigen-antibody reactions are detected by treatment of the strips with antiserum to human IgG (or other globulin) and a specific stain. Molecular weights of the antigenic components are obtained with a pre-stained protein standard (Laemmli 1970) (Fig. 3.5.6). 

The structural proteins of AAV particles have been dissociated with sodium dodecyl sulfate (SDS) or urea and then separated by polyacrylamide gel electrophoresis (Johnson and Hoggan, 1971 Rose et al., 1971). Three proteins were identified with estimated molecular weights of 62-66000 73-80000 and 87-92000, respectively. The smallest polypeptide represented about 80% of the total protein mass while the other two represented approximately 10% each. There was no correspondence in electrophoretic mobility with adenovirus structural proteins except that there was a partial overlap of the major AAV protein and the adenovirus fiber penton. The three AAV capsid proteins were present in all three human serotypes (AAV I-3) and had similar mobilities with the exception that the major protein species of AAV 2 moved slightly faster than the comparable AAV 1 and 3 species. Very minor... 

Detection of CFi subunits. CFi subunits were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose paper essentially as described by Towbin et al. (1979). Specific proteins were detected by the immunoblot assay described by BioRad Laboratories, USA. 

Direct staining of proteins (e. g., after electrophoretic separation in polyacrylamide gels) can be achieved by treatment with dyes like Coomassie Brilliant Blue R-250 [146] (Fig. 7), which binds positively charged proteins in an acidic fixation buffer, allowing detection down to 0.1 pg of protein. 

Determination of Phosphorylation Stoichiometry By biosynthetic labeling, 201, 245 by separation of phosphorylated isoforms, 201, 251 by microchemical determination of phosphate in proteins isolated from polyacrylamide gels, 201, 261 production of phosphorylation state-specific antibodies, 201, 264. 

The IgA banding patterns are seen in the pH 4.5-6.0 region in polyacrylamide gel after protein separation by isoelectric focusing. It was observed in the CSF of multiple sclerosis patients that the intrathecally synthesized viral antibodies of IgG and IgM classes possess oligoclonal characteristics, whereas IgA antibodies appeared to be polyclonal (M3). 

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