A high-yield method for the isolation of hydrophobic proteins and peptides from polyacrylamide gels for protein sequencing.
A methodological approach is described which allows the isolation of hydrophobic and hydrophilic proteins and peptides in high yield. The technique consists of (1) preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) protein elution from polyacrylamide gels with an organic solvent mixture composed of formic acid/acetonitrile/isopropanol/H2O (50/25/15/10, v/v/v/v), and (3) purification of eluted proteins by size exclusion chromatography on a Superose 12 column using this organic solvent mixture as eluant. The efficiency of this technique was tested with radioactively labeled polypeptides. These proteins were reaction center from Chloroflexus aurantiacus, bacteriorhodopsin, halorhodopsin from Halobacterium halobium, bovine serum albumin, ovalbumin, alpha-chymotrypsinogen A, and cytochrome c. The elution recoveries from polyacrylamide gels were 77-95%; the final yield after chromatographic purification was still 67-76% (with one exception). Subsequent amino acid sequencing was possible without further sample treatment. The sensitivity of the method described was found to be at least 20-30 micrograms protein.
Feick
R G
RG
Max-Planck Institut für Biochemie, Martinsried, Bundesrepublik Deutschland.
Shiozawa
J A
JA
D000433:1-Propanol; D000097:Acetonitriles; D000595:Amino Acid Sequence; D001419:Bacteria / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates; D006217:Halobacterium / Q000032:analysis; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis*; D012995:Solubility
D000097:Acetonitriles; D005561:Formates; D008565:Membrane Proteins; C030544:formic acid; D000433:1-Propanol; C032159:acetonitrile