Re: [Histonet] getting rid of biotin-avidin background

From:"Mikael Niku"

Dear Tyrone (and others),

I'm routinely and happily using egg white powder (really raw avidin) and 
biotin (by Sigma) to block endogenous biotin, as suggested earlier on 

Before primary ab (and perhaps also the serum block, doesn't really matter), 
incubate slides for 15 min in 10% (w/v) egg white powder in water.
Wash. Then incubate in 0.1 mg/ml biotin in buffer for another 15 min (it's 
also possible to just add this to the primary ab).

The solutions:

Add 10% (w/v) egg white powder in water.  Stir to solubilize most of the 
stuff. Store in the freezer (seems to be OK to thaw repeatedly). . Before 
use, spin briefly to get rid of the unsolubilized material.

Biotin stock: 1 mg/ml D-biotin in your IHC buffer (you can find the exact 
Sigma product # elsewhere in Histonet, if necessary.. probably doesn't 
matter). Store in the freezer (OK to thaw/refreeze). Dilute 1/10 for use.

Works quite nicely, though I think I never tried commercial blocking kits 
for comparison. I think the crude nature of the "avidin solution" is 
actually a bonus, adding a protein blocking effect.

The most difficult part might be obtaining the egg white powder, which isn't 
available in grocery stores at least in Finland. After some searching, I got 
a huge bag of it from some industrial egg company. Enough for a few 
centuries of use and of course practically free (whereas purified avidin 
seems to be really expensive).

With best regards,

 Mikael Niku, PhD, university lecturer
 University of Helsinki, Division of Nutrition

 - What do I think of western civilization?
   I think it would be a good idea!

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