I have used a wash buffer containing 2% SDS, 4M urea, and 150 mM NaCl for biotin-streptavidin pull-downs. In my experience, this led to decreased non-specific binding with no notable loss of bound biotinylated targets as long as the washes are performed at room temperature.

Dear Rongbing,

As Avidin is a protein, it is also denatured by SDS/Urea. So it is not reusable under harsh conditions. Maybe with mild concentrations it could work, as Biotin/Avidin is one of the strongest binding force in nature. 

You can make SDS agarose - and URIA- agarose gels for electrophoresis - so you can use both

Well it is certainly use urea to wash your agarose beads containing Streptavidin. This is quite standard way to do that. I have my self used streptavidin agarose beads to pull down biotinlylated RNA (Ameta S. and Jäschke A., Chem. Sci., 2013,4, 957-964). 

In fact if you search some papers regarding stability os Streptavidin in urea you will find that indeed Streptavidin can tolerate quite a good concentration of urea. 

So definitely you can use it if your protein doesn't have problems with high urea. Just make sure you have fresh batch of beads else they might bleed-through(some portion of protein will come in your wash fraction). 

Feel free to ask more,

good luck,

Sandeep

I have found several sources that say that once bound, the streptavidin-biotin complex can withstand up to 6M urea and 2% SDS (at room temperature). However, I can't find any sources that indicate whether the presence of these agents in the buffer would prevent the initial binding. Does anyone have experience with that?

Hi, 

In their protocol (https://tools.thermofisher.com/content/sfs/manuals/MAN0011768_ActivX_Serine_Hydrolase_Probes_UG.pdf), Thermo Fisher recommends denaturating Desthiobiotin-labeled samples with a final concentration of 5M urea before capture with streptavidin beads, so I would guess it should work. 

Hello,

Has anyone here tried adding the urea during pull down? What concentration did you use? Did it significantly reduce the amount of non-specific binders while keeping/increasing the number of protein targets bound to the beads?

Thanks!

I have used a wash buffer containing 2% SDS, 4M urea, and 150 mM NaCl for biotin-streptavidin pull-downs. In my experience, this led to decreased non-specific binding with no notable loss of bound biotinylated targets as long as the washes are performed at room temperature.