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?)
