I have a big problem, that is nonspecific binding between my interest protein(desmin) and protein G agarose, protein G sepharose, or protein A agarose when i do the IP assay.
I always blocked 20ul bead each 1.5ml tube with fresh 5%BSA in 500ml PBS overnight before processing IP. Bead is not powder, it's slurry and commercially came from Santa Cruz.
Before Blocking, i washed bead with 1ml PBS for twice from the slurry. Next, taking bead to mix with IGg that can't bind any Ab for at least 1 hour.
When bead is ready, i started the preclering process that puting bead into lysate. The lysate was C2C12 cell and BHK21 line which containing indigenous desmin(intermediate filament),
and centrifugation at 12000rpm for 10 minutes. A lot of desmin exists at pellet, and some of it at supernatant. I took the supernatant for IP assay. Extraction buffer was used for IP buffer, 10mM Tris-HCl PH 8,EDTA 5mM PH 8.08, NaCl 140mM, Triton X-100 1%(or NP-40 1%), protease inhibitor cocktail, phosphatase inhibitor, 2mM PMSF. Sometimes, i added additional 0.1% SDS and 0.5% deoxycholic acid saldium salt as RIPA buffer. Anyway, i always got the same resalt. When 30 minutes preclearing step is done, tube applied to centrifugate at 2500rpm for 3 minutes. I left the supernatnat from pellet, then took it for IP step(Ab 2 hours, bead 2 Hours). The pellet dissolved in sample buffer as the negative control. But negative control always showed that binding between bead and desmin without antibody. I'm trying to Co-IP two proteins, the interacting protein is binding to the agarose G or A beads, so i can't have a negative control.
Any help is appreciated, thanks in advance!
Edited by CCLee49, 08 April 2011 - 12:18 AM.