negative control in IP - (Oct/19/2010 )
hi everybody.
i'm new here, i'm wondering if anyone has an idea to help me.
i'm performing immunoprecipitations of various proteins from cell culture lysate using an A/G protein-agarose resin (santa cruz). i also perform a negative control in which i don't put any antibody at all, but when i proceed through the wester blot, the lane containing the "-Ab" control is still full of proteins. is it possible that the resin itself is binding the whole protein lysate without the Ab (even if i regularly perform a preclearing)? i obviously used a new sample of the resin, in case the old one was contamined with Abs.
any idea?
To begin, this is not the ideal negative control for IPs. You should to be doing an IgG control with normal IgG from the same species that the IP antibody is produced in. Ideally, if you are using a mouse antibody for the IP, your IgG should be the same isotype but this can be difficult so most people just use "normal mouse IgG". Check out santa cruz for these control IgGs. They're really cheap and the most accepted control. Next, in order to prevent nonspecific binding you can try to block your beads before the IP. I incubate my beads in 5%BSA/PBS that has been filtered (this is important!). Although one hour should suffice, I often set my beads up the day before harvesting cells and let them block overnight. You then want to wash your beads 2-3 times to remove all free BSA and proceed to the IP. Do you add antibody to the lysate and then add beads or do you bind the antibody to the bead and then incubate with lysate? Which ever way you normally do the IP, you might want to try the other. I've seen this greatly increase the specific IP so that any background is comparatively decreased.
rkay447 on Wed Oct 20 03:03:44 2010 said:
To begin, this is not the ideal negative control for IPs. You should to be doing an IgG control with normal IgG from the same species that the IP antibody is produced in. Ideally, if you are using a mouse antibody for the IP, your IgG should be the same isotype but this can be difficult so most people just use "normal mouse IgG". Check out santa cruz for these control IgGs. They're really cheap and the most accepted control. Next, in order to prevent nonspecific binding you can try to block your beads before the IP. I incubate my beads in 5%BSA/PBS that has been filtered (this is important!). Although one hour should suffice, I often set my beads up the day before harvesting cells and let them block overnight. You then want to wash your beads 2-3 times to remove all free BSA and proceed to the IP. Do you add antibody to the lysate and then add beads or do you bind the antibody to the bead and then incubate with lysate? Which ever way you normally do the IP, you might want to try the other. I've seen this greatly increase the specific IP so that any background is comparatively decreased.
Thank you for your answer, very useful suggestions. However, I don't really know if it is the same, but the manufacturer says this resin has been pre-blocked with BSA. I'll try anyway. I usally incubate the lysate with Ab O.N. and then I add the resin to the sample. I could try the other way. I still think it's quite odd this resin binds so many proteins without putting any IgG in the sample.
Thanks again