kirby99 Posted January 6 Posted January 6 (edited) Hi, I'm trying to reduce my protein but it seems like its not working. I'm wondering if its a problem with the buffer? I am checking for free cysteines using a dtnb assay, I see reduction after adding >1mM TCEP but it is a very small amount, probably <5% reduction. I am dialyzing too after about 1 hr of TCEP addition... so TCEP is not messing with the dtnb assay... Could there be an interaction between TCEP and tris-hcl pH 7.4 buffer? I've tried another buffer too hepes-nacl ph 7.4 but I dont think that has worked well either. If anyone knows if there is an interaction between tris and TCEP tho that would be helpful... Thanks Edited January 6 by kirby99 Want to be clearer in my question
CharonY Posted January 6 Posted January 6 TCEP is routinely used in Tris under basic conditions. It is less stable in phosphate buffers at neutral Ph. Is your chemical or the solution very old?
kirby99 Posted January 6 Author Posted January 6 (edited) Thanks for responding! I made the proteins in October and purified them through November. I tried reducing them in December and now again in January.... I made a 10 mM stock of TCEP in December in dmso, used it and froze it and used it again in January. The non-dissolved TCEP is from June 2020. My supervisor is thinking that they may have given me the wrong plasmid lol. My protein is supposed to have two cysteines.. I've heard it oxidizes randomly when sitting out, but the TCEP I added should've been enough to reduce it... What could be getting reduced in my mixture though? We purified it with nickel column and anion exchange.. When I run gels of my mixture I only see one band running the correct length... Edited January 6 by kirby99
CharonY Posted January 7 Posted January 7 Not sure without seeing the whole protocol, but from what I see there no urgent red flags. TCEP being a few year old is not ideal, if it was opened years ago and not stored under nitrogen, or at least well-sealed. That being said, I have used fairly old powder without too much trouble (but used it in excess). As you are using Ni-NTA I presume that there are no metal chelators involved. I would probably run the sample through mass spec to see what I got. I am not a big fan of dialysis, as I tended to lose too much. But then that was decades ago, so perhaps the dialysis systems have improved.
kirby99 Posted January 8 Author Posted January 8 I dont think it was stored under nitrogen or really well sealed. I think it was a screw on cap, stored in our regular chest freezer. No metal chelators. Mass spec is an option I guess but at our institution it won't start up for a few weeks. We're sending the plasmid for sequencing this week, so I hope that is the problem. Thanks for your help though! I really thought the problem was going to be in the buffers.
BabcockHall Posted March 18 Posted March 18 (edited) Faucher and Maitre Synthetic Communications 2003 33(20):3503-3511. DOI:10.1081/SCC-120024730 I would consider the possibility that TCEP is reducing DMSO, although the presence of iodine may be necessary for a rapid reaction (see Faucher and Maitre). If I could find no more information on this topic, I would consider switching my solvent to DMF. Is there any reason that you are using TCEP and not 2-ME or DTT as your reducing agent? Edited March 18 by BabcockHall
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