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Is DTT reduction and alkylation critical before concentrating proteins?

IP concentrate mass spec

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#1 liuyi200809

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Posted 15 July 2012 - 02:55 PM

I did an IP experiment and have proteins eluted using 1% SDS/Tris buffer. Usually we use dtt to fully reduce the proteins and perform alkylation for the purpose of mass spectrometry later on. By accident today, I skipped the DTT reduction and also the alkylation step and directly put my IP elution sample into speed vac to concentrate my samples. Though I added DTT after concentrating. I wonder if my eluted proteins will degrade a lot due to be absence of DTT? To what extent would this affect the final mass spec result?

#2 PhDinAcronyms

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Posted 16 July 2012 - 08:45 PM

The DTT is used to reduce any disulfide bonds in your protein and the alkylation is to block the cysteines and prevent them from reforming disulfide bonds. Reducing the disulfide bonds will assist in unfolding the protein so it can be digested by a protease for mass spec (assuming this is the type of protocol you are following). Either way forgetting to add it shouldn't cause your protein to degrade. Whether or not you can add it in out of step will depend on your specific protocol.





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