I am going to purify several proteins by using GFP-nanobodies linked to a sepharose column since my proteins have a GFP taq. Since I have not experience with this protocol, I would like to solve several technical aspects that I do not find them totally clear at bibliography.

1- Can I store my Sepharose columns linked to the GFP-nanobodies for a few months before using them? What is the correct buffer to do so? I have read diverse protocols that use 20% ethanol (in my opinion very heavy to store a protein, but no idea about nanobodies) or NaH3, that I guess is toxic and not very common in the labs. Any suggestion?

2- After purifying my protein and cleave the GFP, it will remain bound to the Sepharose column. Is there any possibility of regenerate the nanobodies by interrupt the Nb-GFP interaction? In this way I would be able to re-use my columns several times.

Thank you very much!

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