2 replies to this topic

[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 and 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 lysate.
Incubate the mixed lysates for 1hr and then add antibody to do Co-ip.

Do you guys consider it is cheating or an acceptable method? (in theory, I think it's acceptable...)

Edited by Jason S LEUNG, 09 January 2011 - 01:14 AM.

[lee_liam]

I also have had this problem exactly. Before I thought it may be the contamination of the empty vector, then I re-maxiprep the plasmid and the situation continued. Now the situation appears sometime only, what I can surely tell you is that once you add MG132, the difference in the expression (A+vec >>> A+B)is even more drastic. Could someone explain that?

[Jason S LEUNG]

lee_liam, on 13 January 2011 - 10:21 AM, said:

I also have had this problem exactly. Before I thought it may be the contamination of the empty vector, then I re-maxiprep the plasmid and the situation continued. Now the situation appears sometime only, what I can surely tell you is that once you add MG132, the difference in the expression (A+vec >>> A+B)is even more drastic. Could someone explain that?

Those seniors in my lab said the up-regulations of co-expressed proteins are commonly seen if two proteins are interacting partners of each others.