IgG purification from hybridomas - (Dec/25/2013 )

Hey everybody, 

I'm trying to purify an antibody from hybridomas. It's IgG and last year i was able to do it on the first time (on G-sepharose beads of SIGMA, o/n at the rate of 0.8ml per min in 4oC) this time nothing is working!!!

the conditions (medium, clone, beads, protocol) are exactly the same but still... Just not working. I already tried protein A beads, pH adjustment with Tris pH=8 (10% added to conditioned medium), changing the sub-clone of the hybridoma--- nothing.  

Does anyone have a piece of advice for me? It's realy frustrating...   

have you tried a fresh batch of g-sepharose beads? the attached protein may have denatured.

Try testing the supernatant (conditioned media) for the presence of antibody then test your flow thru to confirm that it never bound to the column.  Once you confirm that it was there in the first place and never bound then trouble shooting gets a bit easier.