Polyacrylamide gels experimental methods

The molecular weight of the enzyme has remained remarkably constant from the bacteria to the higher mammals (Table VIII). The molecular weight from all species averages 36,500, and the deviations from this value fall within the experimental range of the methods used, in this case sedimentation equilibrium in the ultracentrifuge or electrophoresis of the SDS-enzyme-complexes on polyacrylamide gel. 

In this chapter the experimental practice of analytical polyacrylamide gel electrophoresis will be set out. Analytical methods have been evolved to operate on a scale of say 1-10 /ig or 1000 counts per minute per component, though this can often be appreciably reduced. We shall not concern ourselves here with true micro-techniques, which function down to the level of the contents of a single cell in some cases, since these are of specialised interest, and have been fully dealt with in a recent and authoritative monograph on micro-methods in molecular biology (Neuhoff 1973). 

The experimental details concerning the preparation and characterisations of the catalysts have been described previously [6-7], The main characteristics of the solids are presented in table 1. They were prepared by calcination of polyacrylamide gels in which the metal salts were incorporated. This method allows to obtain the mixed-oxide phases at moderate temperature (TOO C) with rather high surface areas. 

FIGURE 4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) pattern showing the purification of the PSPBP purified from Acanthocardia tuberculatum. Lane 1 and 2 represent respectively crude extract and purified PSPBP fraction. For experimental conditions, see Materials and Methods. 

Protein Adsorption. Since protein adsorption is the earliest event in the thrombotic response of the blood to polymers, the types of proteins adsorbed from plasma to polyacrylamide-Silastic and Silastic tubes were examined. As described in the Experimental section, plasma was recirculated through the tubes for 2 h at 37°C and rinsed away with buffer. The proteins adsorbed to the polymers were eluted with SDS, separated by SDS-polyacrylamide slab gel electrophoresis, and silver-stained using a new extremely sensitive method (19). Figure 10 shows the results of these assays. 

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