on single FLAG tag

[Zhicheng Dong]

Hi All,
I have a problem with detecting FLAG-TAG protein in transgenic plants 
tissue. I tagged my favorite protein, which is 220kD, with FLAG at 
the C-terminus and HA at the N-terminus. I can detect the recombinant 
protein with anti-HA but not with anti-FLAG. I can even pull down the 
tagged protein with anti-FLAG beads. The triple FLAG TAG works quite 
well in our lab. This time I use single FLAG TAG this time because I 
was told it is easier to elute single FLAG tagged protein from the 
anti-FLAG beads than the triple FLAG tagged protein. I have used the 
purified recombinant protein with single FLAG tag to test the 
anti-FLAG antibody. I can hardly detect the 10ng protein with sigma 
M2 anti-FLAG antibody. Did you ever meet this kind of question? Thanks.

ZC