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Protocol for elution of desthiobiotin-labelled proteins from Streptavidin Column

Streptavidin Mass Spectrometry Desthiobiotin Protein purification

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#1 lilpalmitate

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Posted 11 January 2013 - 04:05 PM

Hi all,

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!

LP

#2 neuron

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Posted 17 January 2013 - 08:36 PM

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.





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