Polyacrylamide gel, preparation

R. Azzam, Sandy Soil Plantation in Semi Arid Zones by Polyacrylamide Gel Preparation by Ionizing Radiation, lAEA-Contract No. 2596/RB, Progress Rpt July 1980-Oct. 1981, Vienna. 

Separation Ranges of Polyacrylamide Gels Preparation of Polyacrylamide Gels... 

The leakage of bacteria was minimum when the bacteria were immobilized in polyacrylamide gel prepared from acrylamide containing 10-15 % BIS. Therefore, total acrylamide containing 85 % acrylamide and 15 % BIS was employed for immobilization of the bacteria. 

Hjerten, S. 1962. Molecular sieve chromatography on polyacrylamide gels, prepared according to a simplified method. Arch Biochem Biophys, Suppl 1 147-51. 

The most commonly used combination of chemicals to produce a polyacrylamide gel is acrylamide, bis acrylamide, buffer, ammonium persulfate, and tetramethylenediarnine (TEMED). TEMED and ammonium persulfate are catalysts to the polymerization reaction. The TEMED causes the persulfate to produce free radicals, causing polymerization. Because this is a free-radical driven reaction, the mixture of reagents must be degassed before it is used. The mixture polymerizes quickly after TEMED addition, so it should be poured into the gel-casting apparatus as quickly as possible. Once the gel is poured into a prepared form, a comb can be appHed to the top portion of the gel before polymerization occurs. This comb sets small indentations permanently into the top portion of the gel which can be used to load samples. If the comb is used, samples are then typically mixed with a heavier solution, such as glycerol, before the sample is appHed to the gel, to prevent the sample from dispersing into the reservoir buffer. 

To develop a continuous process, the immobilisation of aminoacylase of Aspergillus oryzae by a variety of methods was studied, for example ionic binding to DEAE-Sephadex, covalent binding to iodo-acetyl cellulose and entrapment in polyacrylamide gel. Ionic binding to DEAE-Sephadex was chosen because the method of preparation was easy, activity was high and stable, and regeneration was possible. 

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

Chromium crosslinked gels prepared from a 3 1 blend of partially hydrolyzed polyacrylamide and guar gum have been found to have a higher strength and stability than gels prepared from the partially hydrolyzed polyacrylamide alone (222). 

There are many examples of ELIS As used for detecting host cell impurities in the literature. Pauly et al.12 developed an ELISA to detect impurities in erythropoietin that had a detection limit of around 0.05 ng/ml. SDS polyacrylamide gel and Western blot analysis were used to confirm the spectrum of proteins detected and to demonstrate the specificity of the antibody preparation. Anicetti et al.14 describe an assay for the detection of E. coli proteins in recombinant DNA-derived human growth hormone. Whitmire and Eaton15 report on an immuno-ligand assay for quantitation of process-specific E. coli host cell contaminant proteins in a recombinant bovine somatotropin. 

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