Electrophoresis in polyacrylamide

Morris, CJOR Morris, P, Molecular-Sieve Chromatography and Electrophoresis in Polyacrylamide Gels, Biochemical Journal 124, 517, 1971. 

Rill, RL Locke, BR Liu, Y Dharia, J Van Winkle, D, Protein Electrophoresis in Polyacrylamide Gels with Templated Pores, Electrophoresis 17, 1304, 1996. 

Mather, I. H., Sullivan, C. H. and Madara, P. J. 1982. Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Biochem. J. 202, 317-323. 

R.T. Swank and K.D. Munkres, Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, Anal. Biochem. 39 (1971) 462-477. 

Immunoblotting techniques involve the identification of a protein target via antigen-antibody-specific reactions. Proteins are typically separated by electrophoresis in polyacrylamide gels, and then transferred ( blotted ) onto chemically resilient membranes (e.g., nitrocellulose, polyvinylidene difluoride) where they bind in the pattern they took in the gel. The membrane is overlaid with a primary antibody directed to the specific target, then with a secondary antibody (anti-immunoglobulin) labeled with radioisotopes, enzymes or other marker compounds. 

Protein samples are separated by one-dimensional SDS-PAGE or two-dimensional gel electrophoresis in polyacrylamide gels. The separated proteins are then transferred (blotted) to a nitrocellulose or nylon sheet. This is incubated with specific antibody to the protein and then unbound antibody is washed away. Those proteins in the gel that bind the antibody are detected either by autoradiography (if the specific antibody was radiolabeled) or by using a second labeled antibody that binds to the primary antibody. 

DNA fragments in a restriction digest can be separated by size by electrophoresis in polyacrylamide or agarose gel. Polyacrylamide gel is used to separate smaller DNA molecules whilst agarose gel has larger pore sizes and so can separate larger DNA fragments. 

Electrophoresis in polyacrylamide gels containing SDS led to an apparent molecular weight of 18,000, consistent with the known amino acid sequence of this protein. Explain these data. 

Total tRNA and 2% tRNA from yeast and bull s liver were obtained according to a previously described procedme [5]. An enriched fraction of specific tRNA was obtained by preparative electrophoresis in polyacrylamide gel [6]. The enriched tRNA preparations contained from 75 to 150 nmoles of tRNA. 

Fig. 6.7. Schematic representation of two types of apparatus for ultramicro-electrophoresis, (a) Ultramicro-electrophoresis on a single fibre of polyacrylamide gel. A, agarose bridges E, electrode vessels F, polyacrylamide gel fibre on the microscope cover glass, (b) Ultramicro-electrophoresis in polyacrylamide gel in a vertical capillary.

Electrophoresis in polyacrylamide gels has been carried out on proteins prelabeled with either fluorescamine (Ragland et a/., 1974) or MDPF (Borges et aL, 1976) (Fig. 2). The influence of pH and acrylamide concentration on the resolution of fluorescamine-modified peptides ranging in size from 3 to... 

High-voltage paper electrophoresis was the first method introduced for the identification of dansylated amino acids and for the separation of peptides [1,6] subsequently, electrophoretic separations on thin layers were also reported [34,35]. These methods are, however, unsuitable for the separation of amine derivatives. TLC on silica gel hamino acid, f>eptide and amine derivatives. A large variety of solvent systems of varying polarity is now available, which allows the separation of even complex mixtures of amino acids [7,36—38] and amines [7,17,39]. Fingerprinting or identification of Dns-peptides can also be carried out on silica gel layers 29,40,41]. For the separation of Dns-peptides, electrophoresis in polyacrylamide gel and on cellogel strips was reconunended (42,43]. Alumina [44] kieselguhr (45] and cellulose thin-layers [46] have also been tried. 

A bbreviations PS - photosystem, RC - reaction center, Chl-pro-tein - chlorophyll-protein, PAGE - electrophoresis in polyacrylamide gel, LHC - light-harvesting complex, SDS - sodium dodecyl sulfate, LiDS - lithium dodecyl sulfate, OGP - octyl- -D-gluco-pyranoside, kDa — kilodalton, P595 - fluorescence with a maximum at 695 nm, Pheo - pheophytin FP - free pigment. 

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