Immunoprecipitation problem - Protein nonspecific binding to bead (Apr/08/2011 )
Hello~ everybody!
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!
You could try increasing the stringence of your IP buffers. Hopefully, the Ab-desmin interaction is stronger than the desmin-bead interaction, so that a higher stringency buffer would eliminate non-specific interactions but still permit the IP. When doing ChIP experiment, we wash with buffers containing up to 500 mM NaCl, and the IP still works.
Hope this helps!
madrius1 on Fri Apr 8 20:04:56 2011 said:
You could try increasing the stringence of your IP buffers. Hopefully, the Ab-desmin interaction is stronger than the desmin-bead interaction, so that a higher stringency buffer would eliminate non-specific interactions but still permit the IP. When doing ChIP experiment, we wash with buffers containing up to 500 mM NaCl, and the IP still works.
Hope this helps!
I appreciate for your help. Do you suggest me that i can increase the salt stringency? Could you tell me why? I put IgG for completing that bead strongly binding to desmin, but still not working.