Mouse IgG1 purification - Protein G - (Aug/15/2012 )
I tried to purify a mouse monoclonal IgG1 from hybridoma supernatant (RPMI + 10% iFBS).
I used HiTrap Protein G column (GE Healthcare). As suggested in the handbook, I used 20mM Phosphate Buffer pH 7.0 as Binding Buffer and 0.1M Glycine pH 2.7 as Elution Buffer. I followed the protocol but I couldn't get any elution peak.
I tested by ELISA the collected flow through samples (loading and washing) and the "elution fractions" but no significant amount of Ab was detected. It means the Ab should be still on the column. I tried to elute even at pH 1 or precipitate it with EtOH but without any success!
Any suggestion for improving the purification? Any suggestion for switching to protein A?
Worst case: how can I "clean" the column?
Thanks for your help! paola
you can try eluting at pH 10.