Preparing samples for SDS gel electrophoresis

Start with a known protein(s). Get the system working with standards before trying unknowns.

Does your sample have enough SDS?
Proteins bind 2x their weight in SDS and lipids (and nonionic detergents) bind 10x their weight. Add increasing amounts of SDS to identical samples and look for trends

Has your sample been heated sufficiently?
I use 3 min at 100C. Heat parallel samples for different lengths of time and look for trends

Are conditions adequately reducing?
Use a difficult to reduce protein such as serum albumin. Compare parallel samples with different levels of reducing agent (ME, DTT)

Is your reducing agent good?
Use different lots, or reduce some of it with a phosphine.

Are your proteins reoxidizing during electrophoresis?
Compare reduced samples to reduced and alklyated. If proteins reoxidize, you can put mercaptopropionic acid (bad odor) in the upper well. This causes "rain" if you stain with silver.

Is the pH of your cracking buffer high enough for good reduction?
Try several pHs. Laemmli gels are insensitive to cracking buffer composition. I use pH ~8.5.

Does your sample have enough protein?
Compare different amounts of sample. If need be concentrate the sample by TCA precipitation or phenol extraction - acetone precipitation

Handling "nasty" samples, i.e crude tissue
Purify proteins away from "junk", i.e. by phenol extraction