Pierce™ Gentle Ag/Ab Elution Buffer, pH 6.6, 3.75 L - FAQs

After immobilizing my antibody, the first affinity purification of my protein worked well, but the protein failed to bind to the antibody during the second round of purification. What did I do wrong?

Low pH elution is the most commonly used method of elution for affinity purification, however, it could lead to denaturation of some antibodies which will negatively affect subsequent antigen binding. To prevent this from happening, after protein purification and column regeneration, immediately wash the column with neutralization or binding buffer and store at 4 degrees C in buffer containing an antimicrobial agent such as sodium azide.

Alternatively, use a near-neutral, high salt elution buffer such as Pierce Gentle Ag/Ab Elution Buffer (Cat. No. 21013, 21027, or 21030). For alternative elution buffers, please refer to this Tech Tip (https://tools.thermofisher.com/content/sfs/brochures/TR0027-Elution-conditions.pdf).

Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.