Fractionation and characterization of an oligomeric series of bovine keratohyalin by polyacrylamide gel electrophoresis

Reevaluation of the Electrophoretic Migration Behavior of Soluble Globular Proteins in the Native and Detergent-Denatured States in Polyacrylamide Gels

“…The demonstration ( (Fig. 5) and in (11,29)) that Ferguson plots yield well-defined K r values, that were T independent and related to protein masses in this range, is in conflict with the prediction by this reptation mechanism ((11), and accompanying data), in which the slopes depend only upon %T and are independent of M r . In an alternative approach for resolving this paradox, absolute R g estimates for SDS-protein complexes were obtained from R plots constructed with the globular protein data.…”

“…Instead, the effective radius (Eq. [3]) for a random coil has been proposed to represent the radius of gyration (R g ), and since R g is proportional to ͌ M r , K r would be proportional to M r , rather than surface area (29). Because of the curvature of K r vs M r plots over large intervals (28; Figs.…”

Analytical and Preparative Polyacrylamide Gel Electrophoresis

“…Addition of SDS to the upper buffer in a concentration below the CMC and one tenth of the buffer constituents (i.e., of the ionic strength of the operative phases) seems as effective as addition of SDS to the gel (M. Wyckoff, D. Rodbard, and A. Chrambach, 1976). Alkylsulfates with an alkyl chain length less than that of SDS (Ugel et al, 1971) appear to have the advantage of relatively easy removal from the protein.…”

“…Thus it is necessary to obtain a Ferguson plot to evaluate whether equal charge density has been achieved. This is indicated by a common y-intercept (or a common point of intersection elose to 0 %T) for the pro teins under study (Ugel et al, 1971;Rodbard, Chapter 3).…”

“…This allows one to derive molecular size data from mobilities at single gel concentrations. The calculation of molecular size for an unknown, by comparison of its R r with a calibration curve based on standard proteins of known molecular weight, should inelude consideration of the fact that the standard curves of log (MW) vs. R f and log (R,) vs. MW are linear only over a very restricted range (Ugel et al, 1971;Rodbard, Chapter 3). Nonlinear curve-fitting methods are therefore required for a generally applicable analysis of these plots (Rodbard,Chapter 3).…”