Applications of tandem microbore liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/electroblotting in microsequence analysis.
Protein isolation by microbore HPLC is compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/electroblotting methods for several major proteins from rabbit muscle. Although single-mode HPLC or SDS-PAGE/electroblotting provides excellent speed and sensitivity for submicrogram-level protein purification, neither one alone has adequate resolution for separating such a complex protein mixture. Tandem procedures, utilizing two different modes of HPLC in separate steps or a combination of single HPLC separation and SDS-PAGE/electroblotting, offer the necessary versatility. One of the major concerns in this investigation was to evaluate electroblotting techniques for microsequencing. The Aebersold et al. procedure (R.H. Aebersold, D.B. Teplow, L.E. Hood, and S.B.H. Kent (1986) J. Biol. Chem. 261, 4229-4238) was substantially modified and improved; the details of this work will be published elsewhere. These changes significantly improve repetitive yields at the low microgram level without producing high backgrounds. At lower levels the recovery of sequenceable protein currently limits our ability to obtain useful results. Starting with 250-750 micrograms of rabbit muscle crude extract, several proteins (15-70 kDa) were isolated by tandem microbore LC and PAGE/electroblotting for amino-terminal sequence analysis. It appears that the combination of electroblotting and microbore LC represents a powerful approach for microsample preparation.
Yuen
S
S
Applied Biosystems Inc., Foster City, California 94404.
Hunkapiller
M W
MW
Wilson
K J
KJ
Yuan
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D008832:Microchemistry; D009124:Muscle Proteins / Q000032:analysis; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate
D009124:Muscle Proteins; D012967:Sodium Dodecyl Sulfate