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!
Frequently, low pH is used to elute from immune affinity columns
e.g. Protein Science 27 (2018) 1083-1092
Article Selectable High-Yield Recombinant Protein Production in Huma...
"Reusability of anti‐GFP/YFP sepharose
The anti‐GFP nanobody binds very tightly to GFP/YFP, and protein cannot be removed using mild conditions. To regenerate the resin after use, we used a low pH wash (0.1 M glycine pH 2) to remove YFP and any remaining uncleaved YFP‐TOP2α from the resin. In this way, we have been able to reuse this resin ∼20 times with no noticeable loss of binding capacity [Fig. 4(I)], similar to other commonly used affinity matrices such as amylose–sepharose, glutathione–sepharose, or Ni–NTA."
"Regeneration of anti‐YFP/GFP sepharose
After TEV elution of TOP2α, resin was washed once with water, three times with three column volumes of 0.1 M glycine pH 2.0, then three column volumes of Lysis buffer. The loss of yellow color served as a visual indicator of YFP removal."
Antibody affinity columns are usually stored in the presence of sodium azide:
https://www.pacificimmunology.com/resources/antibody-characterization/affinity-purification/
"The column should be stored refrigerated in PBS, 0.02% sodium azide. Do not freeze."
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