A simple in situ cyanogen bromide cleavage method to obtain internal amino acid sequence of proteins electroblotted to polyvinyldifluoride membranes.
We report a simple method to obtain internal amino acid sequences from larger proteins electroblotted to polyvinyldifluoride membranes. To demonstrate this method, immunoglobulin heavy and light chains are separated by gel electrophoresis and electroblotted to the membrane. The separated chains, immobilized to the membrane, are cleaved in situ by cyanogen bromide and the resulting fragments are subsequently eluted from the membrane. The fragments are separated by gel electrophoresis, electroblotted and subjected to gas-phase microsequence analysis.
Scott
M G
MG
Department of Pathology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110.
Crimmins
D L
DL
McCourt
D W
DW
Tarrand
J J
JJ
Eyerman
M C
MC
Nahm
M H
MH
D000595:Amino Acid Sequence; D003488:Cyanogen Bromide / Q000494:pharmacology*; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000032:analysis*; D008566:Membranes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping / Q000379:methods*; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D003488:Cyanogen Bromide; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers