two step purification (SPA) concern change to one step? - (May/05/2006 )

I am currently useing SPA purification for protein complexes. so promoter-my protein- camodulin tag-FLAG tag, two step purification, antiFLAG first, then there is a TEV cleavage site in between, then using anti Camodulin. now I found that antiFLAg did not work properly. So I wonered that if i did not cleave off FLAG, directly using anticalmodulin one step purification. is it going to work?
any suggestions? Thank you all so much. here is really important for me for quite a while.

-cathy-

as long as the epitope that the anti-calmodulin is directed against is exposed then you will purify your complex. the tag will still be attached.

-mdfenko-

QUOTE (mdfenko @ May 5 2006, 06:49 AM)

as long as the epitope that the anyi-calmodulin is directed against is exposed then you will purify your complex. the tag will still be attached.

in my case, is it calmodulin exposed if FLAG tag still on? I can not tell.

-cathy-

i would expect some part of the calmodulin to be exposed. i am, unfortunately, not familiar with the flag tag but i don't expect that it would completely envelope the calmodulin.

-mdfenko-

QUOTE (mdfenko @ May 5 2006, 07:31 AM)

i would expect some part of the calmodulin to be exposed. i am, unfortunately, not familiar with the flag tag but i don't expect that it would completely envelope the calmodulin.

i wondered that the antibody conjugated to agarose. agrose is quite big bead, whether agrose can get in?

-cathy-

QUOTE (cathy @ May 8 2006, 10:20 AM)

i wondered that the antibody conjugated to agarose. agrose is quite big bead, whether agrose can get in?

if the anti-calmodulin is conjugated to the agarose then it is attached to the bead, either within the pores and/or on the surface of the bead. the antigen passes through the agarose as it would during gel filtration but gets captured by the bound antibody. the pores in agarose are quite large and can pass megadalton size proteins.

-mdfenko-