Commentary: Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea)

Ksenija Gasig, Schuyler S. Korban

Research output: Contribution to journalReview articlepeer-review

Abstract

Chimeric constructs with the hydrophilic octapeptide FLAG epitope (DYKDDDDK) have been widely used as multipurpose tags for identification, detection, and purification of FLAG fusion proteins. Constructs consisting of C-terminal FLAG-tagged genomic and cDNA clones of an Arabidopsis phytochelatin synthase gene, AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression of AtPCS1 in transgenic lines were confirmed by using PCR and Northern blot analyses. However, immunoblot analysis revealed strong nonspecific binding of a monoclonal anti-FLAG M2 antibody to an endogenous protein in both shoot and leaf tissues of wild-type Indian mustard (85-kDa) that, masked presence of the phytochelatin synthase (PCS) protein of interest (55-kDa). Further analysis revealed absence of a nonspecific protein in root tissues of transgenic plants, thus allowing detection of the FLAG-tagged PCS protein.

Original languageEnglish (US)
Pages (from-to)9-16
Number of pages8
JournalPlant Molecular Biology Reporter
Volume23
Issue number1
DOIs
StatePublished - Mar 2005

Keywords

  • Antibody
  • FLAG peptide
  • Indian mustard
  • Phytochelatin synthase
  • Transgenic plants

ASJC Scopus subject areas

  • Molecular Biology
  • Plant Science

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