I am trying to perform an Immunoprecipitation against FLAG in two different cell lines. When I perform the IP in rescue cell lines (I mean, cells that are overexpressing a construct that contains my gene of interest +FLAG, in KO cells to my gene of interest) it works. The problem appears when I try to immunoprecipitate the FLAG-tagged protein in cells that exhibit a endogenous FLAG-tag system (knock-in cells).
Moreover, when I extract protein from my knock-in cells with RIPA lysis buffer (Santa cruz) I can also detect the FLAG tag. So I supose the problem is due to the low expression of my gene-protein.
In my IP conditions I am using 1 mg of total protein + 2 ug of antibody
How can I improve the IP efficiency?
Hi Vera,
Yes, this mainly would be due to the endogenous protein expression level (driven by it's own promoter rather than an exogenous one like CMV). There isn't much you can do here other than use more protein input, keeping the FLAG-Ab consistant between the lines you are assessing. Essentially, you want to saturate the antibody and beads. Increase your binding time if you can (3+ hours or O/N at 4C). Are you also aiming to identify other proteins attached to your protein (co-IP)? If so, don't use RIPA... it could be too harsh to maintain protein interactions. Instead use a mild lysis buffer. If you are just attempting to see your tagged protein, then it is fine. If you want more info, check out our AP-MS manuscripts and protocols (https://nbcc.lunenfeld.ca/resources/)
Cheers,
Reuben
Nicolas Szabo-Fresnais I was using the promoter of my own gene, in this case is Trdmt1 gene
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