Exclusion limit polyacrylamide gels

Polyacrylamide gels are produced by the copolymerization of acrylamide and the cross-linking agent N,N methylenebisacrylamide. These are supplied by Bio-Rad Laboratories (Bio-Gel P). The Bio-Gel media are available in 10 sizes with exclusion limits ranging from 1800 to 400,000 daltons. Table 3.4 lists the acrylamide gels and their physical properties. 

Fig. 12. Diagram of elution pattern of red cell acid phosphatase and various markers on Biogel P 60. The position of the various protein markers was determined both by optical density determination and by starch gel electrophoresis of the individual fractions (83). The experiment was carried out using a polyacrylamide gel (Biogel P 60, 50-150 mesh exclusion limit >60,000 Bio-Rad Laboratories, California) in 0.05 M tris buffer, pH 8.0, containing 0.08% (v/v) Tween 80 and 0.1% (v/v) 2-mercaptoethanol to stabilize the enzyme. Column 60 X 4 cm. Flow rate 20 ml/hr, 4 ml fractions. (A) OD at 280 nm, ( ) OD at 540 nm, ( ) LDH assay with p-nitrophenyl phosphate for AcP. From Hopkinson and Harris (85).

Gel filtration with polyacrylamide (mol wt exclusion limit from 3000 to 300,000) and agarose gel (mol wt... 

Tier 3 involves the measurement of molecular weight distribution. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) or size exclusion chromatography (SEC) provides information of molecular weight distribution of protein composition on and estimated concentration of proteins >2.5 kDa. Ideally the lower limit detection of the method should be 1 ppm or less based on standard proteins. If the nonfood allergen or... 

Table 5 lists two lines of commercially available silica-based gel permeation columns. The selection was limited to the Zorbax and LiChrospher silicas because these materials were specifically developed for gel permeation chromatography. Zorbax silica has a 6 pm particle size for optimum efficiency. The pore sizes were chosen such that a linear calibration curve is obtained when coupling columns of different pore sizes. In addition to plain silicas, Zorbax silicas are also available derivatized with trimethylchlorosilane, providing a surface that is less adsorptive for certain organic soluble polymers. Several important water-soluble industrial polymers, such as polyacrylamide, polyacrylic acid, and polyvinyl alcohol, do not require deactivation of the silica surface to obtain ideal size exclusion behavior. LiChrospher silicas are 10 pm in size they vary in pore size from 100 to 4000 A to allow the separation of very large polymers. 

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