protocol for membrane receptor isolation

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Thu Jul 3 09:14:53 EST 2008


Am 25.06.2008, 06:18 Uhr, schrieb WS <novalidaddress from nurfuerspam.de>:

> Just wonder if membrane proteins get solubilized without detegent?

No. Membrane attached proteins can be washed of membranes by high salt  
(100 mM Na2CO3 pH 11.5 or 250 mM KI or 2 M NaBr), but transmembrane  
proteins require detergents.

> Will eg 0.5% TX100 disrupt the antibody-recptor complex?

That depends on the nature and concentration of the detergent and on the  
antibody-antigen interactions. Note however that in immunoprecipitation  
studies even relatively harsh detergents like SDS are frequently used to  
ensure specificity. For example, in their booklet on Pansorbin Calbiochem  
recommends the following "superwash buffer": 100 mM NaCl, 50 mM Tris-HCl  
pH 7.4, 2 mM EDTA, 2% Triton X-100, 0.5% SDS. Of course, not all  
antibodies can be used with these conditions, but some titration should  
give you detergent concentrations where specific binding survives with  
minimal background.


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