Phage-display is done using a Triton X-100 cell extract to select our clones. Nanobodies are then produced in the periplasm of E. coli, extracted using B-Per and purified using NiNTA beads exploiting the His-tag of the nanobody. These nanobodies work in IFAs. However, if these nanobodies are no eluted from the beads, but then allowed to bind to proteins of a Triton X-100 cell extract and target proteins eluted with glycine, no specific target can be identified. Can anybody spot any problems with the procedure?
Could you explain a bit more of the details on the elution with glycine step as I think this might be where the issue is. Also could you say how you are trying to identify the target protein is it via mass spec?
Dear Peter,
So we elute with 0.1 M glycine pH 2.7 and immediately neutralize with 1 M Tris-HCl pH8. We then run the samples on a SDS gel and do mass spec on the whole lane. Does this help? I really apprecaite any input!
I would try a Ph2.0 elute. If you do end up the solving the problem please do let me know what solution worked for you as its an interesting problem!
Hi Chandra,
Feel free to forward your question to our tech support team, We have been troubleshooting a wide variety of issues for variety of applications using nanobodies.
Best,
Mary
Hi Peter,
Thank you for the suggestion. We are able to elute the nanobody with pH 2.7, the only problem is that we cannot IP anything with it. Why would you think lowering the pH would help?
Thank you for helping us. I will definitely let you know what happens in future. As this is a low priority project handled by one of our students, it may take some time to get back to you.
Cheers,
Chandra
Dear Mary,
Thank you very much for offering help. I will contact them now.
Kind regards,
Chandra
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