When purifying monoclonal antibodies from hybridome cultures (protein G affinity chromatography) we got two different peaks, which we collected separately. When analysed by SDS-PAGE they both proved to be antibodies (two bands, proper sizes). Have you an explanation for this phenomenon? Could it be something like there was such an antibody excess adsorbed to the column that glycine only released certain amount of antibodies and then, the second peak corresponds to the antobodies that werent accesed by the glycine in the first instance? Do you have any references of that? Thank you in advance
hi
If you are using culture medium containing FBS to produce monoclonal antibodies, try using protein A or Protein L column for mouse IgG purification .
https://www.abcam.com/kits/antibody-binding-affinities-of-protein-a-protein-g-protein-l-and-jacalin
good luck
Amy Mónaco
Could you please show your AKTA SEC image screenshot/image? Have you run the Native-PAGE for both mAb fractions? Which medium was used to grow hybridoma?
Sanjeev Kumar we did not run a native PAGE, why would you do that? What additional info could you gather with this experiment regarding to our question? We used OPTIMEM to induce antibody production, no FBS added.
Find attached a picture of the two peaks
Amy Mónaco
This is useful. Native-PAGE gels of both the protein peak will tell you that whether both peaks are of full IgG 150kDa molecule or mAb is making some aggregates. May be as per stejpan suggestions, you can try using citrate buffer, which is better than Glycine buffer.
Hope this is useful.
Hi Amy Mónaco,
I agree with the above and suggest you try citrate buffer.
Good luck!
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