1 reply to this topic

[RCholic]

Hi,

I use 293T cells for co-transfection and my proteins of interest are overexpressed well in the transfeced cells. I usually do 48 hr transfection and I can get about 2 mg of whole cell extracts from the cells (in a volume of 350 ul with RIPA lysis buffer).

However, I wonder what's the minimal amount of cell extracts is required for a successful Co-IP (my proteins of interest have good overexpression).

Thanks!

[bob1]

RCholic, on Jul 6 2009, 10:12 AM, said:

Hi,

I use 293T cells for co-transfection and my proteins of interest are overexpressed well in the transfeced cells. I usually do 48 hr transfection and I can get about 2 mg of whole cell extracts from the cells (in a volume of 350 ul with RIPA lysis buffer).

However, I wonder what's the minimal amount of cell extracts is required for a successful Co-IP (my proteins of interest have good overexpression).

Thanks!

The answer depends on your antibody and the interaction between your protein of interest and the target protein.  Weak interaction and low affinity antibody, and you will need lots of protein.  Strong interaction and high affinity antibody and you won't need much protein.