Isolation of low picomole levels of peptides and proteins for microsequence analysis: An overview

The ability to purify and manipulate low picomole levels of peptides and proteins is now considered to be the rate-limiting step in obtaining amino acid sequence data from such low-abundance samples. Typical final high-resolution purification techniques include RP-HPLC, affinity chromatography and polyacrylamide gel electrophoresis. In this article we report two strategies for sequencing two-dimensional polyacrylamide gel (2-DE) resolved proteins. In one strategy, proteins were visualized in the gel by staining with Coomassie blue and then recovered by electroelutioru In the other strategy, proteins were visualized in the gel by high-resolution dynamic imaging and then passively eluted. For microsequencing, proteins were isolated from the electroeluate or passive eluate, free of SDS and gel-related contaminants, by “inverse-gradient” RP-HPLC.