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Protocol: Explanation of Our Sample Preparation Alternatives

Choice of Rehydration Buffers

DeStreak Rehydration Solution

This general purpose solution, which contains urea, thiourea, CHAPS and the disulfide exchange reagent DeStreak, is a wonderful general purpose rehydration buffer. It is almost always used when the proteins analyzed are soluble or loosely held in membranes.

Bio-Rad Sequential Extraction Reagent 3

This solution contains 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10, 40 mM Tris and ampholytes. The SB 3-10 in this mixture is a stronger detergent than CHAPS and helps solubilize intrinsic membrane proteins. This rehydration buffer is recommended when the sample contains membrane proteins.

Another rehydration option for membrane proteins is 7M urea 2m thiourea, 2% ASB-14.

Two-step Sample Preparation

If a customer wishes to view all proteins in a cell or tissue, it is strongly recommended that the sample first be separated into soluble and membrane-bound components. To do this, the sample should be initially homogenized in a gentle non-detergent buffer. The homogenate is then separated into soluble and membrane-bound fractions by centrifugal methods. The proteins in the soluble supernatant are precipitated and dissolved in DeStreak Rehydration Solution. The membrane pellet is washed well and then usually dissolved in Bio-Rad Sequential Extraction Reagent 3. All of the proteins may be recombined so that they are run on one gel by dissolving the precipitated soluble protein pellet in Bio-Rad Sequential Extraction Reagent 3 and then using this solution to dissolve the membrane pellet.