HELP!!! How much cell extract to use in CO-IPs with HA-mAb?
ashaw5 at aol.com
Sun Apr 23 16:14:10 EST 1995
I think the easiest way to think about this is to try and calculate the
number of expressed molecules you are getting in your Cos cells and then
try and figure out what concentration of the protein you will need to
detect the interaction assuming your interaction has a respectable Kd (say
10-100nm). For now, I think that the Western is the best place to start.
It is the least confusing and the most convincing if your experiment
works. But it depends on the sensitivity of your Western. Again you
should be able to calculate the sensitivity of your Western and then
figure how much of your protein needs to be expressed and co-precipitated
for you to detect it. If you have too much background, try changing the
detergent and preclearing your IP's. Also remember that sometimes
co-expressing the two plasmids in the same cells works much better and
sometimes is required to see the interaction (in the case that the
assembly of the complex is regulated by the cell). Good luck.
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