FLAG-tagged protein expression problem

[Yenching]

Dear colleagues,

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 
in potato.



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.



Yunching



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