2 replies to this topic

[Opal]

I'm doing a co-IP and I wanna first make sure that I pull down enough of my protein  . So when I do a western blot and compare the protein in my total lysate with the IP it looks like that I'm only precipitating about %2 of the protein (comparing a %2.5 input with the total output). I'm using an anti-serum which is not purified but is highly specific for my protein. Does any body know if this yield is normal? which steps I should work on to increase the yield? adding more serum to my tissue lysate? and/or adding more sepharose beads? it's also worth mentioning that most of the protein remains in the supernatant after the incubation with antiserum and the beads!

Thanks

[Arun Kumaran]

I got ~0.3% only from plant tissue. It doesn't matter for me since it is enough for further process. Try to use more beads.

[Opal]

Thanks Arun,
I've already increased the amount of beads so right now I'm adding ~50 ul of slurry beads to 140 ul of lysate which has a total protein concentration of ~15 ug/ul. I'm afraid if I increase the beads more than this or if I add more of the antiserum used for IP I will increase the non-specific pull down. currently all of the proteins which I'm testing for Co-IP are also present in the IP using preimmune serum