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 anArabidopsis phytochelatin synthase gene,AtPCS1, were used in developing transgenic lines of Indian mustard. Presence and expression ofAtPCS1 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.
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Abbreviations
- PC:
-
phytochelatin
- PCS:
-
phytochelatin synthase
- cDNA:
-
complementary DNA
- gDNA:
-
genomic DNA
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Gasic, K., Korban, S.S. Nonspecific binding of monoclonal anti-FLAG M2 antibody in Indian mustard (Brassica juncea). Plant Mol Biol Rep 23, 9–16 (2005). https://doi.org/10.1007/BF02772643
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DOI: https://doi.org/10.1007/BF02772643