Please comment on this Co-ip method

[Jason S. LEUNG]

I'm a newbie of doing co-ip

 

I always find it's very difficult to adjust similar co-transfected vs single transfected protein expression amount for co-ip several different constructs. (My boss's absolute requirement before actually doing co-IPs) like:

 

Protein A + vec

Protein B + vec

Protein C + vec

A+B (both upregulated)

A+C (both upregulated, but A has much more upregulated level than A+C or A alone)

 

So, the level of A will never be the same in all cases (A, A+B, A+C)!

 

One of my labmates suggests me to simply mix single protein-transfected HEK293 lysates together.

i.e. A-transfected lysate+ B-transfected lysate = A+B. Do it for all sorts of combination

Incubate the mixed lysates for 1hr and then add antibody for 4hr for Co-ip.

Do you guys consider it is a cheating or an acceptable method?

(In theory, I think it's acceptable...rite?)

Edited January 11, 2011 by Jason S. LEUNG