FLAG-tagged protein expression problem
hnbjed at yahoo.com
Tue Jan 25 19:35:40 EST 2005
I constructed a FLAG tagged vector with FLAG at the N-terminal (using
pCAMBIA 1303) for protein purification. I cloned my gene into it, PCR
FLAG-gene and cloned into pBS to sequence the gene, and determined it
is in frame with the FLAG epitope. I transformed WT Arabidopsis with
my construct. I verified the existence of FLAG-gene in the transgenic
genome using PCR.
Recently, I did Western blot using anti-FLAG monoclonal M2 antibody
from Sigma and also used positive control bought from Sigma
(FLAG-tagged BAP protein). Unfortunately I did not see expression of
my fusion protein, but the positive control showed a single band in
the membrane. Interestedly, I observed cross reaction of anti-FLAG
monoclonal M2 antibody with one large protein from WT Arabidopsis,
which was ever reported by Korth et al(2000) Plant J. 23(4), 507-516
Need I do RT-PCR to verify that the FLAG-tagged gene is transcribed?
Can someone give me advice on how to tackle my problem?
Thanks a lot.
E-mail: hnbjed at yahoo.com
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