Hi,

Try to use Glycine 100mM, pH 2.5 -3.0 plus EDTA 0.1 -0.3M. Also you can try to elute by competition using FLAG peptide in a neutral buffer (the same binding buffer). Maybe you can try to elute at alkaline pH around 10-11, Ihad an experience with a MAb with high Kaf.

Good luck!!

Hello. In my experience boiling in 1x laemmli buffer might be the only way to actually get nearly 100% of your protein eluted. Because that method is unacceptable for you, have you considered the following:

What kind of volume are you using for glycine or flag peptide? If the volume is too small, perhaps not all the beads are immersed in solution leading to poor elution. You could try a larger volume of the same concentration of glycine/peptide then spin concentrating back down later.

Alternatively, you could try increasing the concentration of Flag peptide. More peptide should mean a higher probability of it outcompeting your protein off the beads. I know Sigma usually says 100 ug/mL is sufficient but if your resin is saturated, you could try 250-500 ug/mL and see if this helps your recovery.

Also, I usually elute with peptide for an hour on the rotator. It could be that you simply not giving the elution enough time to occur.

Maybe a combination of the above factors.

Dear Kaumeel,

Sorry to hear about your challenges.

Ab affinity

The success of elution depends in our experience very much on the affinity of immobilized antibody and the captured target. For strong binders it can be very challenging to elute with high efficiency with mild elution methods (high salt or low pH as tested here).

Elution volume

Our experience when optimizing both manual and automated (KingFisher Flex) IP protocols for the Dynabeads also showed that the elution volume is one of the critical parameters. An elution volume as low as 20 µL resulted in 30 % less yield compared to 100 µL elution volume. The elution volume used will of course be a trade off between yield and the usability downstream (e.g. volume of the wells in the gel for electrophoresis. - we have used the Bolt gels with the capacity of up to 40-60 µL/well). The smaller the volume the harder it is to work with as well (not sure which beads are in use in these experiments). Too many beads in combination with high amount of target in a small volume may also have an impact on the elution efficiency.

Kind regards

ketil

Commercial anti-FLAG beads are optimized for low-pH elution, so you should not need to use high-pH (aminomethylpropanol) or chaotrope (guanidine). I recommend rinsing the beads in dH2O before elution, to remove residual load/wash buffer. Or increase volume of elution buffer to 200 uL for 50 uL beads. These steps will confirm the beads reach the target elution pH.

If your tagged protein is hydrophobic, it may stick to the beads non-specifically after elution. For this problem try including 0.1% Tween 20 in the elution buffer.

Dear Oscar Otero Alfaro,

Thank you very much for your suggestions. I have been only using Glycine-HCl, without EDTA. Maybe in the next experiment, I will try elution with Glycine-HCl + EDTA.

Unfortunately, the FLAG peptide too doesn't work so effectively.

Dear Evan Kerek

Thank you for the suggestions. I always make sure that beads are immersed in the elution solution. Generally, for 50 ul beads (i.e., 25ul net), I use 50 or 75 ul of elution solution. For 100 ul beads (i.e., 50 ul net), I usually use 100 ul of the solution. I do not think volume is the issue in this case.

I have also tried a higher concentration of FLAG peptide with a longer time. It has improved the elution, but not so satisfactorily.

Dear Ketil Winther Pedersen,

Thank you for answering my question. I don't think elution volume is the problem as I always make sure that the beads are well immersed in the solution.

I think the challenge is due to a strong binding between the Ab and the target.