Peptide mapping and internal sequencing of proteins electroblotted from two-dimensional gels onto polyvinylidene difluoride membranes. A chromatographic procedure for separating proteins from detergents.
Direct sequence analysis of proteins electroblotted from two-dimensional polyacrylamide gels onto immobilizing matrices provides an efficient technique for obtaining N-terminal sequence data for proteins not amenable to purification by reversed-phase high-performance liquid chromatography (RP-HPLC). We present in this paper a procedure for obtaining peptide fragments from electroblotted proteins for internal amino acid sequence analysis. First, Coomassie Blue-stained proteins are extracted from polydivinylidene difluoride membranes, using a detergent mixture of sodium dodecylsulfate and Triton X-100. Proteins are then separated from the detergent mixture by a chromatographic procedure which relies on the ability of proteins to interact with certain reversed-phase sorbents at high organic solvent concentrations. Under these conditions, detergents and Coomassie Blue are not retained and pass through the column. Proteins are recovered by simultaneously: (i) introducing trifluoroacetic acid into the mobile phase and (ii) decreasing the organic solvent concentration. After proteolytic fragmentation, peptides are purified by microbore-column (1-2 mm I.D.) RP-HPLC for microsequence analysis.
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research (Melbourne Branch), Victoria, Australia.
R J
RJ
Simpson
L D
LD
Ward
G E
GE
Reid
M P
MP
Batterham
R L
RL
Moritz
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000032:analysis; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification
D000596:Amino Acids; D003902:Detergents; D006023:Glycoproteins; D008567:Membranes, Artificial; D011506:Proteins