I would like to identify a calpain cut site in a 190 kDa protein. My plan is to express a c-terminally tagged protein, purify it by IP, and then conduct an on-bead digestion. Once digested, the sample will be sent for sequencing by Edman degradation to identify the cut site(s). Does this sound like an appropriate strategy? What source(s) of recombinant calpain are recommended? And what buffers are best? Cheers.
Dear Michael,
We have performed a successful analysis and identification of calpain cleavage sites within the Machado-Joseph disease protein ataxin-3. Our investigations were mainly based on recombinant purified ataxin-3 which was incubated with commercially available recombinant purified calpain-1 and calpain-2. The identification of cleavage sites was performed using a SILAC / mass spectrometry-based methodology. A detailed description can be found in our paper: https://academic.oup.com/brain/article/140/5/1280/3063601
If you need further information, don't hesitate to contact me directly.
Best,
Jonasz
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