Micropreparative protein isolation from polyacrylamide gels following detection by high-resolution dynamic imaging: application to microsequencing.
Polyacrylamide gel electrophoresis is a high-resolving technique for separating proteins. Until recently, its use as a micro-preparative method for isolating microgram amounts of protein in a form suitable for structural analysis was limited by the difficulty in recovering material from the polyacrylamide matrix. This problem has been largely overcome by the development of chemically inert membranes that retain proteins after electroblotting from the polyacrylamide gel. The immobilized proteins are suitable for chemical identification by direct microsequence analysis. Nevertheless, the overall recoveries of proteins using this electrotransfer/blotting approach are low (28%-30%). This paper describes a phase-contrast technique that allows the visualization of proteins during the polyacrylamide gel electrophoresis. This approach is suitable for the quantitative detection of proteins. Since no fixing/staining step is employed, proteins can be recovered in high yield (50%-65%), by passive elution, in a form suitable for microsequence analysis. Methods and strategies for obtaining internal amino acid sequence information from proteins recovered from gels after visualization by high-resolution dynamic imaging are discussed.
Ward
L D
LD
Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Parkville, Victoria, Australia.
Simpson
R J
RJ
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D007782:Lactoglobulins / Q000302:isolation & purification; D008832:Microchemistry / Q000379:methods; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D014357:Trypsin
D007782:Lactoglobulins; D011506:Proteins; D014357:Trypsin