Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing.
A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.
Liang
S P
SP
Department of Chemistry, Boston University, Massachusetts 02215.
Lee
T T
TT
Laursen
R A
RA
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005898:Glass; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D013861:Thiocyanates
D011506:Proteins; D013861:Thiocyanates; D012967:Sodium Dodecyl Sulfate; C032582:bitoscanate