Support polyacrylamide gel

Abraham etol. [43] suggested the use of polyacrylamide gels supported in microporous membranes. This approach could provide the packaging advantages... 

Polyacrylamide gel is the most commonly used type of support medium for gel electrophoresis, and polyacrylamide gel electrophoresis is simply known as PAGE. The gel is usually formed by polymerization of acrylamide and the cross-linking agent N, iV -methylene-bis-acrylamide (Bis) in the presence of ammonium persulfate (APS, initiator) and N, N, N, iV -tetramethyl-ethylenediamine (TEMED, accelerator). The total concentration of acrylamide... 

Zonal techniques are the most frequently used form of electrophoresis and involve the application of a sample as a small zone to a relatively large area of inert supporting medium which enables the subsequent detection of the separated sample zones. A wide range of supporting media have been developed either to eliminate difficulties caused by some media (e.g. the adsorptive effects of paper) or to offer additional features (e.g. the molecular sieving effects of polyacrylamide gel). 

A partially purified HIV viral lysate is laid onto a sodium dodecyl sulfate (SDS)-polyacrylamide gel slab and then electrophoresed, which distributes the HIV peptides through the gel by their relative molecular mass. The higher-molecular-mass proteins form bands near the top of the gel. The proteins on the gel are then transferred electrophoretically onto nitrocellulose paper. The paper is sliced into thin strips, each having the full distribution of HIV antigen bands. The strip is used as a solid support of an indirect immunoassay, and antigen-antibody reactions form insoluble colored bands on the strip. 

In a second step, the gel is fimctionahzed for NA attachment. Common methods for polyacrylamide gel fimctionahzation are based on the treatment of the polymerized support with reagents such as hydrazine or ethylenedi-amine. These treatments generate amine groups in the gel that can react with amine-modified ONDs via glutaraldehyde coupling, or directly with oxidized DNA probes (Fig. 15). Alternatively, the fimctional groups may be introduced by copolymerization reactions (e.g. co-polymerization with N-hydroxysuccinimide acryhc or oxirane acryhc derivatives) [59]. 

HDL, respectively. By starch block, some of the VLDL exhibit 0 2-mobility, whereas by polyacrylamide gel electrophoresis the pre-j8 band migrates in post-)8 position, a phenomenon due to the sieving effect of the supporting medium. 

To illustrate the diversity of strategies available for solid-phase synthesis, several fairly recent protein syntheses can be cited. SRY, an 80-residue DNA binding protein, was syn-thesized[62] on a Pepsyn support (polyacrylamide gel beads) in a continuous flow machine using Fmoc/tBu protection and TBTU/HOBt activation. HPLC gave a homogeneous product with molecular weight 10051 Da (calculated 10033 Da). It bound to DNA, as expected. 

Multipoint attachment to a support protects the enzyme from inactivation by organic solvents. Mozhaev et al. (1990) have recently demonstrated that covalent linkage to polyacrylamide gel stabilizes df-chymotrypsin from denatmation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. 

Polj/meric supports based on polyacrylamide are s)mthesized by copol3tmerization of acrylamide and a cross-linking reagent and can be used directly in affinity chromatography due to its more hydrophilic properties than polystyrene supports. Polyacrylamide gels are either soft... 

Four methods have been developed for enzyme immobilization (1) physical adsorption onto an inert, insoluble, solid support such as a polymer (2) chemical covalent attachment to an insoluble polymeric support (3) encapsulation within a membranous microsphere such as a liposome and (4) entrapment within a gel matrix. The choice of immobilization method is dependent on several factors, including the enzyme used, the process to be carried out, and the reaction conditions. In this experiment, an enzyme, horseradish peroxidase (donor H202 oxidoreductase EC 1.11.1.7), will be imprisoned within a polyacrylamide gel matrix. This method of entrapment has been chosen because it is rapid, inexpensive, and allows kinetic characterization of the immobilized enzyme. Immobilized peroxidase catalyzes a reaction that has commercial potential and interest, the reductive cleavage of hydrogen peroxide, H202, by an electron donor, AH2 ... 

Support Protocol 2 Silver Staining of Polyacrylamide Gels B3.1.15... 

Zone electrophoresis is normally carried out horizontally in a suitable medium such as paper, polyacrylamide gel, starch gel or cellulose acetate. The sample components can be completely separated and quantitatively and qualitatively identified in much lower quantities than by the moving-boundary method. The procedure consists of saturating the support material with a buffer solution. The ends of the strip of support are immersed in separate reservoirs of buffer solution to maintain the saturation. The sample is then applied as a narrow band near one end of the support strip. A voltage potential is created down the length of the strip causing the sample components to ionize and then migrate at a rate dependent on their charge, molecular size and interactions with the support medium. When the process is complete, the strip is removed and developed for examination of the separated components. Densitometry is normally used for quantitation of the bands after suitable color development. 

Zone electrophoresis is used mainly as an analytical technique and, to a lesser extent, for small-scale preparative separations. The main applications are in the biochemical and clinical fields, particularly in the study of protein mixtures. Like chromatography, zone electrophoresis is mainly a practical subject, and the most important advances have involved improvements in experimental technique and the introduction and development of a range of suitable supporting media. Much of the earlier work involved the use of filter paper as the supporting medium however, in recent years filter paper has been somewhat superseded by other materials, such as cellulose acetate, starch gel and polyacrylamide gel, which permit sharper separations. 

Because GFPuv exhibits stability to extreme conditions such as exposure to heat and chemical denaturants (disinfectants) in a wide pH range, its expression by prokaryotes, followed by extraction and purification, should be studied for its potential utility as a marker in validation procedures. In addition, the protein extracted from E. coli and further purified by hydrophobic interaction chromatography (HIC) resins should be analyzed qualitatively (2) by sodium dodecylsulfate polyacrylamide gel (SDS-PAGE) to define the best purification method. SDS-PAGE with Coomassie or silver staining provides a sensitive method to determine the most appropriate HIC support for the purification of GFPuv. 

This is a composite support consisting of polyacrylamide gel trapped in the porous structure ofkieselguhr. The loading is typically 0.1-0.2 mmol amine/g resin [17—19],... 

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