Dear Community,
we have been struggling this year with purification of some antibodies from serum samples and or HEK293T/Recombinant produced antibodies. We have attempted ProteinG, Affigel-peptide, Affigel-protein, Econopac system (from Biorad, based on DEAE ionic exchange) with only 2 successes (from more than 50 attempts). Most protocols (but econopac, that requires change of buffer before DEAE) start from the original buffer conditions, then elutes with Acid Glycine and neutralizes immediately in phosphate buffer pH8.
Please help. Any help (detailed protocol, small talk, instructions, SOP) would be really appreciated.
Cheers,
Mariana
Dear Mariana,
We used to purify antibodies in our lab from various sample sources, including serum, hybridoma culture supernatant, and ascites fluid. I feel this process is greatly aided by the use of purification kits and columns. I would recommend trying a purification kit, such as the Protein A Antibody Purification Kit (ab288103), which has proven to be highly efficient in our work.
Warm regards,
Ishrat
Hi Mariana,
Sounds like your antibody is acid labile.
You could use Pierce Gentle Elution Buffer (https://www.thermofisher.com/order/catalog/product/21027) or you could make your own, see Harlow and Lane - Antibodies.
3.6 M MgCl2
Best wishes
Stan
Thank you both for your
answer.
I will try both ideas in the coming month.
Best.
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