Hi all. I am doing a digestion assay with trypsin and I obtained a protein fragment which resist the trypsin cuts. I want determine the sequence of this fragment for doing a construct. Which technique is better for that?
Thank you
Hi there,
MS analysis of the isolated fragment should be enough to get its sequence.
Hi David, if you have the isolated fragment, you can do MS like Dominique suggested.
If you don't have access to MS, you can reduce and alkylate the fragment, run it on a gel, cut out the band and do old school protein sequencing.
Thank you for your answers. But doing MS is very difficult because to do that technique you have to have the protein pure and When I do the trysinization the protein isn't pure and I will couldn't do other purification step because I will lost much protein.
Steingrimur, yes I did N-term sequenciation but the result is my protein doesnt cut to N-term.
Thank you
Dear David
It is remarked that trypsin cuts partially C-ter of alkalin amino-acid as Arg and Lys. After trypsin action if need to determine sequence of fragment , you can use derivated method with OPA, PITC, Fmoc followed by separation HPLC inverse phase.
My contribution
Marius
Hi David, I don't understand. You have a trypsin protein fragment that has a blocked N-terminus?
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