Does anyone have a protocol/tips for cross linking whole rat sciatic nerve for subsequent coIP?

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Mohammed Hakim Jafferali

You can try DSP crosslinker of 1mM concentration and crosslinking time 15 or 30 mins. it os cleavable with DTT.

DSP crosslinks primary amines, Since proteins are enriched in primary amine, it should be possible for your study. You can check this article where I used crosslinker for CoIP.

http://www.sciencedirect.com/science/article/pii/S000527361400217X

Corey Heffernan

Thanks Mohammad. I have used DSP quite regularly. I think I need another approach. Thanks for your reply!

Oscar Ramirez

Have you tried UV crosslinking?

Thanks for your reply, Oscar. As I understand UV X-linking, I would need to transfect photoactivatable amino acids to the target cells for incorporation into translated proteins - thus, allowing for X-linking. However, I wish to X-link extracted sciatic nerve from rats. Since I don't culture the extracted nerves, simply going straight to X-link and extraction, I am not sure the UV approach is feasible in my system. If I mis-understand the procedure, please do correct me. Thank you again for your reply.

Lena Saal-Bauernschubert

Hi Corey, did you already find any solution for your question? I'm just interested because at the moment I'm facing the same problem. Is it possible to crosslink the whole nerve tissue and go directly for co-IP? Although I'm working with mouse at the moment, but should work the same as for rats.