A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range.
We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.
Institut für Biochemie, Medizinische Fakultät, Universität zu Köln.
J
J
Kurth
W
W
Stoffel
D000595:Amino Acid Sequence; D000818:Animals; D002021:Buffers; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008832:Microchemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification*; D011506:Proteins / Q000302:isolation & purification*; D013108:Sphingomyelin Phosphodiesterase / Q000302:isolation & purification; D014907:Whales
D002021:Buffers; D009211:Myoglobin; D010455:Peptides; D011506:Proteins; D013108:Sphingomyelin Phosphodiesterase