I have been doing affinity purification for a few months now, everything was working fine before but I am getting no protein with the newly packed columns. Antibody coupling efficiency is above 80% and I have used 0.1 M acetic acid (pH 2.4) for elution, I don't know what s going wrong and looking for an opinion if there is a way to check antibodies are intact? Please comment if you think there could be another issue
Thanks
1. test your passed through solvent when loading samples to make sure that no sample is trapped in the column. 2. Use another batch of antibody. 3. make sure that your sample doesn't denature. 4. check the solvent you used to prepare sample solution. 5. check if you used the right resin.
Hi there,
Your elution buffer is not actually a buffer as pH 2.4 (by the way I have in mind that 0.1M acetic acid gives a pH around 2.9) is far from acetic acid pKa (which is around 4.8).Therefore the pH may be affected when in contact of the column matrix. For optimizing elution please read the following from ThermoFisher:
https://tools.thermofisher.com/content/sfs/brochures/TR0027-Elution-conditions.pdf
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