How to improve yield of IP - (Mar/15/2005 )

I want to ip a protein for an enzymatic assay.
I'm using RIPA buffer and HeLa cells as a source. The IP works in general, but i get only a small amount of protein.

I'm using 2µg of rabbit-IgG and 1ml cell lysate with about 3µg/ml, incubate 2h 4°C and pull down with A/G sepharose beads.

But even if i use more concentrated lysate or double the amount of AB, I don't get more IPed protein.

How can I improve the yield? Use even more AB or lysate or should i dilute the lysate?

Someone any suggestions?

thx
Pit

Hi
increase the concentration of the antibody
incubate overnight with shaking,
diluting your cell lysate is a good choice.
why don't your do a titration experiment using different amount of cell lysate with a constant amount of anitbody, then see whats the minimum required for precipitation
Oh and add more beads, incubate for longer time with the beads.
be careful when you wash
good luck

thanks,
i'll try this