Identification of plant mitochondrial proteins: A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a fastblot cycle

Bryan Dunbar, Thomas E. Elthon, John C. Osterman, Beth A. Whitaker, S. Brian Wilson

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Identification of the 329 spots visible in 2D gels of plant mitochondrial proteins is a challenge. This paper describes a 2D mini-gel protocol involving free-radical scavengers and purified reagents to make it compatible with protein sequencing, and evaluates its performance. The paper also describes a "FastBlot" sequencing cycle with the cycle time for protein sequencing from PVDF membranes reduced to less than 29 min with femtomole sensitivity. Other benefits of the cycle include reduced lag, reduced background, reduced loss of labile residues, and increased initial and repetitive yields. The procedure gave excellent results with maize mitochondrial proteins: of six protein spots that we tried to sequence, only one was blocked. The other spots yielded considerable sequence information. One spot was identified from the sequence as superoxide dismutase, while another spot corresponded to an unidentified cDNA from rice. The results of these experiments show that modifications of our previous procedures can provide good N-terminal protein sequencing from individual spots on 2D gels. The technique makes it possible to obtain sequence data, prepare gene probes, and identify many of the polypeptides in the 2D-gel map for plant mitochondria.

Original languageEnglish (US)
Pages (from-to)46-61
Number of pages16
JournalPlant Molecular Biology Reporter
Volume15
Issue number1
DOIs
StatePublished - Mar 1997

Fingerprint

Plant Proteins
Mitochondrial Proteins
two-dimensional gel electrophoresis
Protein Sequence Analysis
Electrophoresis, Gel, Two-Dimensional
Staphylococcal Protein A
Gels
Membranes
gels
proteins
Free Radical Scavengers
Superoxide Dismutase
Zea mays
Mitochondria
Complementary DNA
free radical scavengers
Peptides
polyvinylidene fluoride
plant identification
polypeptides

Keywords

  • Plant mitochondria
  • Protein sequencing
  • Two-dimensional gels

ASJC Scopus subject areas

  • Molecular Biology
  • Plant Science

Cite this

Identification of plant mitochondrial proteins : A procedure linking two-dimensional gel electrophoresis to protein sequencing from PVDF membranes using a fastblot cycle. / Dunbar, Bryan; Elthon, Thomas E.; Osterman, John C.; Whitaker, Beth A.; Wilson, S. Brian.

In: Plant Molecular Biology Reporter, Vol. 15, No. 1, 03.1997, p. 46-61.

Research output: Contribution to journalArticle

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