Desalting Electroeluted Proteins with Hydrophilic Interaction Chromatography

✍ Scribed by P. Jeno; P.E. Scherer; U. Manningkrieg; M. Horst

• Book ID: 102967596
• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 592 KB
• Volume: 215
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1993.1589

✦ Synopsis

We describe a chromatographic procedure for the removal of sodium dodecyl sulfate (SDS) from proteins isolated by electroelution. It involves chromatography of electroeluates on poly(2-hydroxyethyl-aspartamide) silica, a support initially developed for hydrophilic interaction chromatography. The electroeluate, dialyzed against ammonium bicarbonate-SDS buffer, is directly injected onto the column, which is equilibrated in an (n)-propanol concentration greater than (60 %). Bound proteins are eluted with a gradient of decreasing (n)-propanol. This procedure removes essentially all of the Coomassie blue-related contaminants and separates SDS from the protein. Due to the use of volatile buffer systems, the proteins are recovered in completely saltfree form, which facilitates further protein manipulation. After removal of the organic solvent from the chromatographic desalting step, the recovered proteins are directly amenable to (\mathrm{N})-terminal protein sequencing and, after evaporation of the organic phase, to enzymatic digestions and subsequent separation of fragments by reverse-phase HPLC. 1993 Academic Press, Inc.