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Elution of biotonylated proteins

DTT Biotin

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#1 jameshall1384

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Posted 18 September 2013 - 05:11 AM

Hey all,

 

I would like to elute a biotinylated protein sample from strepavidin beads in a way that is compaticle with mass spec. I will use the EZ Biotin with the S-S link. I see that I basically have two options: 1) elute in a DTT in SDS elution buffer and then run in a gel propr to in-gel digestion, but this will lose a lot of sample

 

or

 

2) do an 'on-bead' trypsination i.e. don't elute at all, however I understand this can give a lot of false positives

 

I don't understand however, why I cannot simply elute from the beads with DTT in water. Is the S-S link not supposed to provide a method where you can elute with DTT and thus reduce the S-S bond without the harsh conditions (i.e. SDS) required for disrupting the strong bond between biotin from streptavidin. I must be missing something (or just being plain stupid).

 

Any advice will be greatfullt recieved

 

JH

 

 



#2 sheldon

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Posted 24 February 2015 - 12:55 PM

In terms of eluting biotinylated proteins, if the resin contains a streptavidin variant that releases biotin more readily, this would solve your problem. For example, we made affinity resin with monomeric streptavidin and used it to capture biotinylated GFP. The bound protein can be eluted with free biotin, not unlike elution of 6xhis tag containing protein with imidazole.  

 

A caveat is that the affinity of monomeric streptavidin is less than that of wt streptavidin. So if the protein is present at a very low concentration, the capture efficiency would be low. 

 

Here's our website, in case this helps: http://www.acsu.buffalo.edu/~sjpark6/

 

 






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