Common Artifacts and Mistakes Made in Electrophoresis

Abstract

Proteases that act at room temperature upon proteins in the sample buffer prior to heating, cleavage of the Asp–Pro bond upon prolonged heating of proteins at high temperatures, contamination of sample or sample buffer with keratin, leaching of chemicals from disposable plasticware, contamination of urea with ammonium cyanate are some subtle artifacts that can have significant deleterious effects on carefully planned and executed experiments. In addition, researchers are culpable of committing mistakes with respect to (a) calculating the cross-linking factor of a gel, (b) polymerization temperature and time for a polyacrylamide gel, (c) inducing aggregates in samples for electrophoresis, (d) titrating the running buffer in electrophoresis, (e) proper sample preparation, (f) amount of protein to be loaded on a gel, (g) sample buffer-to-protein ratios, (h) incompletely removing phosphate buffered saline from cells prior to cell lysis and (i) overfocusing of IPG strip in two-dimensional gel electrophoresis. Taking proper heed to all these factors can greatly help generate perfect experimental results.