Quantification of Proteins by Staining in Polyacrylamide Gels
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Quantification of proteins is a common challenge. There are various methods described for estimation of the amount of protein present in a sample as described in Chapters 1–9. These quantify total protein present, but do not quantify one protein in a mixture of several. For this, the mixture must be resolved. Liquid chromatography achieves this, and the various proteins may be quantified by their absorbance at 220 or 280 nm. Microgram to submicrogram amounts of protein can be analyzed in this way, using microbore HPLC. Problems may occur if particular species chromatograph poorly (such as hydrophobic polypeptides on reverse-phase chromatography). An alternative is polyacrylamide gel electrophoresis (PAGE) as the mixture-resolving step, followed by protein staining and densitometry. Exceptions are small peptides that are not successfully resolved and stained in gels. Microgram to submicrogram amounts of protein (of over a few kilodaltons in size) may be quantified in this way. Although not every protein stain is best suited to this purpose, the method described herein is suitable for quantification of microgram-to-submicrogram amounts of proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels (1,2).
KeywordsSodium Dodecyl Sulfate Zinc Salt Reactive Blue Standard Curve Equation Stain Protein Band