Weird IP results! - (Apr/05/2005 )

i'm having trouble with my negative control in ip experiments... here's the set up:

tube 1. antibody and lysate
tube 2. lysate alone

incubate o/n in the cold room; add beads and incubate for 4 hrs. wash 4x with 1ml lysis buffer. elute, run gel, western blot with same antibody as ip (i don't have another functional antibody).

my problem is that i detect my protein in tube 2, which is just the lysate and the beads. the band is about 50% weaker than what i get for the ip... but it's still there. i use proteinG beads from amersham. i've done ips before with these beads and this never happened. could it be that my protein is really sticking to the beads??? i didn't think this could happen.

thanks for the help,
edlira

Hi edlira,

You will find that protein will stick to the Protein G sepharose beads. I always find that a pre-clearing step is desirable where you incubate your lysates with beads for approximately an hour, spin down discard the beads and perform your IP on the supernatant. Obviously the best preclearing is using the Protein-G beads however obviously this is expensive, an alternative is to use the same matrix ie. Sepharose 4B beads I've found this works just as well.

Hope this helps

Scott

that makes sense.. i'll try that next. thanks for your help scott