A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes.
A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.
Warlow
R S
RS
Immunology Department, Repatriation General Hospital, Sydney, N.S.W., Australia.
Gooley
A
A
Rajasekariah
P
P
Oszarac
N
N
Walls
R S
RS
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005782:Gels; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011145:Polyvinyls; D011506:Proteins / Q000737:chemistry*; D011968:Receptors, Histamine / Q000032:analysis; D017421:Sequence Analysis / Q000379:methods*; D013194:Staining and Labeling; D005465:Fluorocarbon Polymers
D005782:Gels; D008567:Membranes, Artificial; D010455:Peptides; D011145:Polyvinyls; D011506:Proteins; D011968:Receptors, Histamine; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers