SDS is a powerful detergent, which will solubilize many cells and tissues. This greatly facilitates sample preparation for SDS PAGE because most samples will be completely dissolved by heating to 95°C in loading buffer (detailed below). A somewhat stronger loading buffer, containing more SDS and Dithiothreitol (DTT) at a higher pH, can be used for the homogenization of more difficult samples.
In general, the goal of sample preparation is to denature the proteins fully, to disrupt any disulfide bonds through reduction, and to dissolve any particles which would interfere with electrophoresis. Incomplete denaturation will not fully saturate the proteins with SDS and will lead to blurred bands or altered mobilities. Failure to dissolve sample particulate completely will result in clogging of the gel which will cause streaking from the well to the end of the gel. Disulfide reduction is often required to release subunits from multimeric proteins. In many instances, it is instructive to run samples with and without reduction, to demonstrate which bands are released by disulfide disruption.
Sample Preparation for SDS-PAGE Electrophoresis
Mix sample with an equal volume of 2X sample buffer (For greater reproducibility, employ National Diagnostics preformulated 2X sample buffer, Protein Loading Buffer Blue 2X). Bring to 95° C for 10 minutes, cool to room temperature before loading. If particulate is present, centrifuge samples 5 minutes at 14k RPM in microcentrifuge, and load the gel. This protocol is sufficient for most tissue culture cells, fluid samples (such as serum and cerebrospinal fluid), bacteria and some soft tissues. For more highly structured samples, use the following buffer:
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CHES Sample Buffer:
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1% CHES pH to 9.5 with NaOH
2% SDS
1% DTT
10% glycerol
Store @ -20° C up to 6 months.
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Homogenize samples in 5 - 15 volume of CHES buffer in a dounce homogenizer @ RT, 25 - 50 strokes.
Heat homogenate to 95°C for 5 minutes and allow to cool. (homogenized samples will contain substantial amounts of cellular debris, which must be removed by centrifugation to avoid clogged wells. Centrifuge samples @ 14K RPM in microcentrifuge, 15 minutes.
NOTES ON YEAST AND BACTERIA: these organisms may be encapsulated in a layer of lipopolysaccharide (LPS) which will require enzymatic digestion with Lysozyme or Zymolyase prior to homogenization.
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