Antibody Purification Troubleshooting




Antibody has eluted in the wash step (not bound)

Incorrect AbSelect™ resin selected for the antibody species

Ensure you select an AbSelect™ kit that is suitable for the host species of your antibody. We have three types of AbSelect™ kit (three different resins): the original AbSelect™ uses Protein A (e.g. high affinity for rabbit IgG); AbSelect™ G is based on Protein G (e.g. use for sheep, goat, rat and human); and AbSelect™ Mouse contains a proprietary resin specific for mouse IgGs (so is not suitable for other species).

Actual antibody concentration much lower than expected; contaminant proteins are what has eluted in the wash step

If the starting antibody concentration had been measured by an absorbance reading, this could have actually reflected a high concentration of other proteins, which will not bind to the resin and will be collected in the wash fractions; and if the true amount of antibody is very low, it will still be in the eluted fractions but its concentration may be too low to be measurable. Please note the minimum antibody amount stated on the protocol for the particular AbSelect™ kit you are using.

More Resources:

Antibody Purification Illustrated Assay