Membrane protein prep

[Ole Diettrich]

On Sat, 15 Mar 1997, Shinhan Shiu wrote:

> Dear bionetters,
> 
> I am trying to purify a plant transmembrane receptor. When I go through the 
> literature, some groups advise aganist the use of boiling in loading buffer 
> prior SDS-PAGE for membrane protein. 
> 
> I wonder if you have experience with membrane protein and found this to be 
> true.
>
>  
> 
> 
> *********************************************
> Shinhan Shiu
> Department of Botany University of Wisconsin-Madison
> 132 Birge 430 Lincoln Dr, Madison, WI 53706
> tel: 608-262-4008
> sshiu at students.wisc.edu
> *********************************************


Dear Shinhan Shiu,
I have not observed any problems with boiling membrane proteins in 
loading buffer and never heard of anything similar. But I observed better 
separated and sharper protein bands in silver stained gels when I was 
using a loading buffer with an additional 6 M urea in it and instead of 
boiling I was just incubating the sample at 37 C for 5 min. Another 
point, which may be especially important with membrane proteins, is the 
necessity to add the boiling loading buffer quickly to your sample, because 
heating the mixture slowly, could result in partial denaturation of some 
proteins exposing protease sensitive domains, which could be digested 
by proteases that are still active under the prevailing SDS and 
Temperature conditions. This may sound like a purely academic question, 
but it actually happened to a collegue of mine, who took quite some time 
finding the problem.
Greetings,
Ole

Dr. Ole Diettrich
Postdoctoral Research Assistant
Institute of Child Health
University College London