GAL4 Nuclear Localization Signal

[George Theodoris]

Neigeborn, Lenore (neigeborn at OCELOT.RUTGERS.EDU) wrote:
: Hi folks,
:      This is in response to George's concern that his protein may not be
: functional when fused to the GAL4-DNA Binding Domain.  He wants to do a
: complementation test with his fusion protein to confirm that his protein is
: still functional (and presumably retains the ability to associate with its
: normal partners).  Right, George? 
Yes. 
:      My question is this:  Have you tried the complementation test yet?

Yes, and it does not complement. Actually I have 2 genes fused to the 
GAL4 DNA binding domain that I am getting ready to screen for interacting 
proteins. Both are cytoplasmic. I have only tested one of them for 
complementation and it did not complement. I still need to test the other 
one. This protein is known to dimerize in the cytoplasm. I fused it to 
both the activation and DNA binding vectors, and it did not interact with 
itself in the 2-hybrid system. So I'm a little worried about the ability 
of these proteins to associate with their normal partners when fused to 
GAL4 and dragged into the nucleus. I think I will go ahead and screen the 
library since I have the plasmids and library ready to go.
						-George