Hello,
I followed a certain protocol on reducing and alkylating proteins in solutions and applied it to on-bead reduction/alkylation with my proteins captured on Neutravidin resin. I was wondering if excess DTT (~60 mM) and iodoacetamide (200 mM) since this what I have used. I eventually found a paper that used less than 10 mM for each reagent. Has anyone have an idea if excess of these reagents would denature streptavidin and release my captured proteins?
Thanks!
It is very likely. By using such a high concentration of reducing agents, you will be getting reactions that simply don't occur at lower concentrations.
Hi Kiall, I would be more concerned about the high iodoacetamide conc. because it can react with amino terminii, Lys, His, and Cys.
Basic questions:
Does non-engineered strepavidin actually have any disulfides and therefore reducible by the DTT? Are you using strepavidin or Neutravidin because both are stated in your questioning? Neutravidin is de-glycosylated avidin while strepavidin is a different protein from Streptomycetes. Both have extremely high affinity for biotin; I recall ~10 exp-14 but strepavidin has higher affinity for conjugated biotin whether as the non-covalent homo-tetramer or monomer. Are you referring rather to reduction of the biotin conjugated target in which case the biotin would still likely be available for binding?
Hey all,
Thank you all for responding. I split my beads with the bound proteins into two, half of them without reduction/alkylation while the other was subjected to the conditions stated above. I did the tryptic digest on-bead as well. Indeed, the concentration of the peptides in the reduced/alkylated proteins on beads was like 3-fold in excess compared to the not reduced/alkylated one (they should have roughly the same concentrations). Obviously, avidin molecules were presumably released from the beads. I tried out these conditions since I was worried that incomplete alkylation of Cys in my pulled down proteins would affect the MS proteomic analysis (since Cys should be set to static modification). The method I initially found made no mention about the total amount of proteins/beads used in the on-bead reduction/alkylation process, that is why I resorted into this what turned out to be a really harsh condition. I am planning on trying out lesser concentrations of DTT (~15 mM) and iodoacetamide (~25 mM) and shorter heating/reaction time. Again, thank you all for your responses!
PS. I used Neutravidin, which I hoped to be more stable and robust under these conditions but it turned out it could not handle it. Lol.
It's now clearer what you are trying to do; didn't know you were trying on-column protease digestion. So it is your biotin conjugated protein that is subject to reduction/alkylation and not the avidin, right? What would happen if you tried reduction/alkylation of the Neutravidin resin and then see if your biotin conjugated protein would still bind?
Hi Grant,
It appears to me that resin-bound avidin in the Neutravidin beads were released and digested, owing to the increased concentration of the tryptic-digested peptides. I decided not to run it on MS to save money and time. I have this strong feeling that my sample is dominated by avidin, which will only muffle my proteins of interest for identification. I will keep this post updated once I am able to do the on-bead reduction/alkylation. Currently, I ran out of my sample. Lol. Thank you!
Indeed, the avidin was likely digested by the protease; probably not unexpected. Did you check the amino acid sequence of avidin to see if it contains any cysteines that can be reduced/alkylated?
Hello again,
When you include the sequence of avidin in the database search, which particular FASTA sequence should be used? I was trying to look for a UniProt ID that Thermo based the structure of avidin from but to no avail. There are several avidin isoforms in UniProt and I can't seem to decide which ones to include. Should I rather include all of them? Haha.
Thanks,
Kiall
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