Applications of an automated apparatus for two-dimensional electrophoresis, Model TEP-1, for microsequence analyses of proteins.
Two-dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two-dimensional electrophoresis, Model TEP-1, which allows precise analytical runs under computer-control with a fully automated transfer from the first- to the second-dimensional gel. In the present paper we describe several applications of TEP-1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP-1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP-1, followed by blotting, provides spots containing ca. 10-100 pmol protein. These figures permitted the identification of 10-40 amino acid residues from the N-terminus. The TEP-1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.
Nokihara
K
K
Tokyo University of Agriculture and Technology, Japan.
Morita
N
N
Kuriki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation*; D005069:Evaluation Studies as Topic; D008049:Lipase / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009154:Mutation; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
D001426:Bacterial Proteins; D011506:Proteins; D008049:Lipase