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sodium dodecyl sulfate-proteins can be stacked in sharp

Time:2015/11/28 5:44:03

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: Properties of the stack, valid Rf? measurement, and optimized procedure

sodium dodecyl sulfate-proteins can be stacked in sharp starting zones in sodium dodecyl sulfate-PAGE in multiphasic buffer systems, Stacking of sodium dodecyl sulfate-proteins has been possible with a lower stacking limit of up to 0.300, above neutral pH in “nonrestrictive” gels.

Under the same conditions, sodium dodecyl sulfate is stacked. Stacking of sodium dodecyl sulfate, derived from both the sodium dodecyl sulfate content of the sample and the upper buffer, broadens the stack in proportion to sodium dodecyl sulfate load and duration of electrophoresis. Such broadening produces an inordinately wide starting zone in the stacking gel, leading to impaired resolution, unless one limits the sodium dodecyl sulfate load. In the resolving gel, this zone broadening makes the trailing edge of the stack, and the tracking dye migrating there [e.g., bromphenol blue at low gel concentration (%T)] unsuitable as a valid reference for Rf. However, the leading edge of the stack and the tracking dye migrating there (e.g., pyronin-Y-sodium dodecyl sulfate at low %T) are a valid Rf reference. At high %T, both sodium dodecyl sulfate and pyronin-Y-sodium dodecyl sulfate unstack, whereas bromphenol blue now moves to the leading edge of the stack and becomes, for that %T range, a valid reference of Rf.

An optimized system for sodium dodecyl sulfate-PAGE is described which contains no sodium dodecyl sulfate in the gel, 0.03% sodium dodecyl sulfate in the upper buffer, 150 μg or less sodium dodecyl sulfate in the sample, and a single set of gel buffer constituents in stacking and resolving gels.

Valid Rf measurement is a requisite for linear Ferguson plots (log Rf vs %T). Since the slope of these plots, KR, is a measure of molecular weight the procedure of sodium dodecyl sulfate-PAGE proposed in this study should provide improved reliability of melecular weight estimates.