TY - JOUR
T1 - Desalting electroeluted proteins with hydrophilic interaction chromatography
AU - Jeno, P.
AU - Scherer, P. E.
AU - Manningkrieg, U.
AU - Horst, M.
PY - 1993/12
Y1 - 1993/12
N2 - 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 salt-free form, which facilitates further protein manipulation. After removal of the organic solvent from the chromatographic desalting step, the recovered proteins are directly amenable to N-terminal protein sequencing and, after evaporation of the organic phase, to enzymatic digestions and subsequent separation of fragments by reverse-phase HPLC.
AB - 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 salt-free form, which facilitates further protein manipulation. After removal of the organic solvent from the chromatographic desalting step, the recovered proteins are directly amenable to N-terminal protein sequencing and, after evaporation of the organic phase, to enzymatic digestions and subsequent separation of fragments by reverse-phase HPLC.
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U2 - 10.1006/abio.1993.1589
DO - 10.1006/abio.1993.1589
M3 - Article
C2 - 8122792
AN - SCOPUS:0027372411
SN - 0003-2697
VL - 215
SP - 292
EP - 298
JO - Analytical biochemistry
JF - Analytical biochemistry
IS - 2
ER -