nonspecific bands after co-ip - (Sep/03/2009 )
Hello, i'm studying interaction between two proteins using co-ip from mouse brain. i have met with some difficulties and i don't know what to do. need help as i am at a loss 
i use same antibodies for IP and the subsequent WB, thus i see the heavy and light chain of the antibodies in the western. but that is not my problem, since the proteins i'm looking for are 37 and 42kD, so the antibody doesn't bother me. the problem is that i see nonspecific bands just in the area of 40-42kD, exactly where one of my proteins is and i can't distinguish the bands, it's more like a smear in that exact area. so i don't know if i have coip or not... i have run just the antibody on a gel (added sample buffer and cetra...), transfered it and western bloted it just as i do my coip. instead of seeing just the two expected bands, i saw a smear around the area of 40-42kD. could it be that the antibody itself has degradated or something? what are these bands? has anyone seen this? is there a way around it?
thanks, alot in advance
bandc on Sep 3 2009, 05:19 AM said:
i use same antibodies for IP and the subsequent WB, thus i see the heavy and light chain of the antibodies in the western. but that is not my problem, since the proteins i'm looking for are 37 and 42kD, so the antibody doesn't bother me. the problem is that i see nonspecific bands just in the area of 40-42kD, exactly where one of my proteins is and i can't distinguish the bands, it's more like a smear in that exact area. so i don't know if i have coip or not... i have run just the antibody on a gel (added sample buffer and cetra...), transfered it and western bloted it just as i do my coip. instead of seeing just the two expected bands, i saw a smear around the area of 40-42kD. could it be that the antibody itself has degradated or something? what are these bands? has anyone seen this? is there a way around it?
thanks, alot in advance
It is generally recommended that one use different antibodies for an IP and the subsequent Western blot to reduce non-specific binding. Try a different antibody. Also, several companies market IgG "cleanup" kits to perform IPs without seeing the heavy/light chains, you may want to take a look at these as well.
Dr Teeth on Sep 3 2009, 03:01 PM said:
bandc on Sep 3 2009, 05:19 AM said:
i use same antibodies for IP and the subsequent WB, thus i see the heavy and light chain of the antibodies in the western. but that is not my problem, since the proteins i'm looking for are 37 and 42kD, so the antibody doesn't bother me. the problem is that i see nonspecific bands just in the area of 40-42kD, exactly where one of my proteins is and i can't distinguish the bands, it's more like a smear in that exact area. so i don't know if i have coip or not... i have run just the antibody on a gel (added sample buffer and cetra...), transfered it and western bloted it just as i do my coip. instead of seeing just the two expected bands, i saw a smear around the area of 40-42kD. could it be that the antibody itself has degradated or something? what are these bands? has anyone seen this? is there a way around it?
thanks, alot in advance
It is generally recommended that one use different antibodies for an IP and the subsequent Western blot to reduce non-specific binding. Try a different antibody. Also, several companies market IgG "cleanup" kits to perform IPs without seeing the heavy/light chains, you may want to take a look at these as well.
hi, and thanks for the reply.
the problem i'm facing is probably not the heavy/light chains as the smear that i see is lower in size.
of course it would be better to use different antibodies, but i don't have that possibility.
the question is what is it that i'm seeing in the size 40-42kD? could it be degradation products of the antibody? is it something that usually is seen when doing IP and WB with same antibody?