Biotin/streptavidin/blocking

Dr. David Martin david.martin at biotek.uio.no
Thu Dec 5 11:22:17 EST 1996


In article <199612051520.JAA05199 at borcim.wustl.edu>,
   brett at BORCIM.WUSTL.EDU (brett) wrote:
+>>Fellow researchers:
+>>I've boitinylated cultured cells, lysed with 2X SDS gel loading 
buffer with 
+>>BME, sheared DNA, ran on SDS PAGE, transfered to PVDF, blocked with 
5% 
+>>milk/TBS+0.05% tween 20, probed with streptavidin-HRP conjugate and 
+>>detected with ECL detection solutions 1+2, home made but good:-)
+>>I would like to know what is the best blocking solution for this 
procedure?
+>
+>I've used 3% BSA *or* 5% nonfat dry milk in TBS-tween, with 
equivalent results.
+>The BSA costs more, though.
+>

0.25% gelatin in 50mM tris pH 7.4 150mM NaCl 5mM EDTA .1% Triton X-100 
also works fine. Another thing I find helps is to wash twice without 
detergent/block just prior to adding the ECL reagent.

.d

* David Martin - Atherosclerosis and Thrombosis research group  *
* http://www.uio.no/~damartin/      david.martin at biotek.uio.no  *
* Arbeid. 22 95 84 54 Telefax. 22 69 41 30  Mobil. 90 74 27 65  *



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