Immunoprecipitation---Why would a 250kDa band be present for the purification of - (Feb/14/2012 )

Help!!
I have been trying to purify out a 25kDa protein from a tissue sample and I have gotten no bands present around that molecular weight. I am however, getting a strong band at 250kDa for my positive and negative control. I am using IgG Agarose beads from an IP kit...I have tried changing the concentration of the protein I am attemping to purify, with no change in my results.

What can I change to get some sort of result to show that my protein is being purified out?? I have been working on this technique for almost a year now and have gotten no viable results.

I might be able to help. I did IP for a long time. you must not get 250 KDa band, that's weird, although I also had non specific bands after purification. However my purified band was way stronger.

do you use antibodies from different species for IP and wb? it sounds like that, because if you use same species your protein of interest will fall behind the lower chain of IP antibody.

can you post your protocol? and what lysis buffer do you use? how do you lyse?

I am using the same primary antibody for both IP and WB.... My protocol is from Sigma-Aldrich...for IgG Agarose beads using spin columns. I use a T-Per lysis buffer when I lyse the cells before I homogenize them...the homogenate is kept in a -20 degree freezer...I also you a protease inhibitor to prevent protein degradation prior to electrophoresis via SDS-PAGE.

Run Ouchterlony on all abs with your protein to confirm binding is taking place.

I have performed numerous slot blots and to determine whether or not the antibody is binding to the protein of interest. Each of those resulted in a strong signal for the that protein.

last time I wanted to detect active Bax and I used 3xT75 flasks. maybe you need a bigger cell pellet. I don't know what protein you wish to purify.

Aquaporin-2 protein

so maybe you need more cells

I am using up to 500 microliters of cells for the IP, what would cause a lack of signal, even though I am using more cells for an IP than would use if I was performing just a western blot?