For those who have conducted Co-IP before, I would like to know the amount of protein required for Co-IP experiment. We will use lysate from cells.
Hi:
It's dependent on your immunoprecipitants, so there is no exact answer. However, if the Co-IP is for overexpressed protein, 500 ug will be enough. For endogenouse protein, it is better at least double (1 mg), but it is still dependent on the expression level of the protein you want to detect. So, please check the expression level of your target proteins before you start Co-IP. Moreover, antibody is also critical for Co-IP, you need to check it, too.
Hi there,
I agree with the above answer, 500µg is the range I consider for 50µL samples. So basically cell lysate protein content of 10µg/µL is OK.
As Chao suggested, it depends a lot on the proteins you are trying to IP. The proteins study are very abundant in most of cell lines. Therefore, I prefer somewhere between 50-100 microgram for IP
However, majority of proteins do not have that high expression in cells. Therefore, 500-1000 microgram will be good starting point if you do not know the expression level of your protein of interest.
Thank you all for your help! Do you think the amounts should change if I'm looking for lipid raft proteins?
Hi:
What kind of lipid raft proteins do you want to examine? Do you want to purify endogenous or overexpression protein? Your question is not clear. However, If you use whole cell lysate, it is better to use at least 1 to 2 mg protein for CoIP. To be honest, it is better to check the expression level of your target proteins before you performing CoIP.
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