Protocol for elution of desthiobiotin-labelled proteins from Streptavidin Column - (Jan/11/2013 )
Long time lurker - this forum has helped me out from time to time so I figured to try my luck here!
I am looking to set up a proteomics experiment - Long story short, I am planning to tag proteins of interest with desthiobiotin then binding them to streptavidin. I would like to elute my proteins using free biotin, but I've yet to find a detailed protocol. Does anyone have a tried-and-true elution protocol that they'd like to share? (ie. how much biotin, buffer composition, etc?), or any other suggestions? My wash buffer and samples are going to be PBS-based.
I am avoiding elution by boiling in SDS buffer to minimize streptavidin contaminants for the MS. While I could do an on-bead trypsin digest to bypass the elution issue, PEG contaminants have been a big problem in our lab and we can't track down the source, so it was suggested that we should elute our proteins and run them into SDS-PAGE followed by in-gel digestions to get rid of the PEG.
Also, if I elute using biotin, should I do an extra desalting step to remove the free biotins?
Thank you guys so much!
I have done this kind of work but the difference is, my biotin was attached to small molecule and I was trying to find a binding protein. I struggled a lot. There is no fixed protocol per se. I used various buffers to standardize and for me 10mM HEPES worked best. For elution I used mild detergents. If ypu are going to elute with excess biotin, then it would be good to run on the gel no? you will get rid of biotin. I don't know how much desalting will help in this case.
A bit of a late reply, but... I was considering using desthiobiotin before. There is that one paper suggesting its use that I'm sure you've seen, but I found the technical bullet for Sigma product 'Desthiobiotin PEO iodoacetamide' to be useful.
Also, what is the advantage of using the desthio for you? Do you want to exclude endogenously biotinylation proteins, or is it for an easy elution? Cleavable biotinylated tags are another option.
Can you digest on-bead? That appears to work well.