Deblocking and Subsequent Microsequence Analysis of N.ALPHA.-Blocked Proteins Electroblotted onto PVDF Membrane.

• Hirano Hisashi

  Institute for Protein Research, Osaka University

• Komatsu Setsuko

  Department of Molecular Biology, National Institute of Agrobiological Resources

• Takakura Hikaru

  Institute for Protein Research, Osaka University

• Sakiyama Fumio

  Institute for Protein Research, Osaka University

• Tsunasawa Susumu

  Institute for Protein Research, Osaka University

抄録

A method was developed for direct microsequencing of N^α-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N^α-Acetylated proteins (>32pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37°C for 30min. The protein was digested on the membrane with 5-10μg of trypsin at 37°C for 24h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50mU of acylamino acid-releasing enzyme at 37°C for 12h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.

収録刊行物

• The Journal of Biochemistry

  The Journal of Biochemistry 111 (6), 754-757, 1992

  The Japanese Biochemical Society

詳細情報

• CRID: 1570854178139593856
• NII論文ID: 130003417432
• ISSN: 0021924X
• Web Site: https://jlc.jst.go.jp/DN/JALC/00078657663?from=CiNii
• 本文言語コード: en
• データソース種別:
  • CiNii Articles