High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane.
In this report we describe the use of a novel, experimental, polyvinylidene fluoride-based membrane with a cationic surface for the isolation by electroblotting of small amounts of proteins separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis. The membrane is characterized by a surface that mediates primarily ionic protein/membrane interactions and that allows the recovery of adsorbed proteins at high yields under relatively mild conditions. In electroblotting experiments, the novel membrane has a binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted proteins in situ. Intact electroblotted proteins, or fragments thereof, were eluted at high yields. Further structural analysis is demonstrated using reverse-phase high-performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed-liquid- or solid-phase automated sequence analysis.
Patterson
S D
SD
Cold Spring Harbor Laboratory, New York.
Hess
D
D
Yungwirth
T
T
Aebersold
R
R
D002412:Cations; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008567:Membranes, Artificial; D010434:Pepsin A / Q000378:metabolism; D011145:Polyvinyls; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012862:Skatole / Q000031:analogs & derivatives / Q000737:chemistry; D013194:Staining and Labeling; D005465:Fluorocarbon Polymers
D002412:Cations; D002918:Chymotrypsin; D003488:Cyanogen Bromide; D008567:Membranes, Artificial; D010434:Pepsin A; D011145:Polyvinyls; D011506:Proteins; D012862:Skatole; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C005904:BNPS-skatole