I am basically working with self-assembled VLPs. I am not sure if His tag in my protein is not exposed (I can detect in reducing western blot by his tag antibody) or other technical issues, and most of my protein appear in flow through. I used pH of 8 for buffer. Should I wash with several column volume of buffer before starting? Thank you for any other suggestions!
DEar Rui Wu
if you are working with VLP is it higly probable that the His-tag is not exposed on the VLP surface.
Are you using His tag fused at N-term or C-term? or did you tried both solutions? Do you have some spacers beetween your protein and the His-tag?
best
Manuele
Yes, washing with several column volumes of buffer before loading the sample is a good idea to equilibrate the resin. Other suggestions:
pH 8 is fine, but ensure it doesn’t exceed 8.0. If binding is weak, try lowering the pH slightly (7.4-7.6).
Keep the imidazole concentration low (~10-20 mM) during the binding phase to reduce non-specific interactions but allow proper binding.
Allow the sample more time to bind to the resin (30-60 min at 4°C).
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