Protein preps from tumor tissue: need help.

[Dr E. Buxbaum]

sundares at bcm.tmc.edu (Sundaresan Venkatachalam) wrote:
>
>ear Netters:
 I am unable to get a protocol (from thr regular methods books)
>that would allow me to run a SDS-PAGE + Western using tumor tissue. In
>addition is it necessary to do an immunoprecipitation  when dealing with
>tissues?  I would appreciate if someone can give me some pointers/
>references for my questions. Thanks a lot.

Why should you have to use different procedures for tumor and normal 
tissue? The method depends more on the properties of your protein than 
the source of tissue. Is it soluble or membrane bound (in the latter case 
you have to include detergent)? Does it tend to aggregate (then you use 
chaotropic agents like urea)? Is it a very rare protein? Then it might 
pay to isolate the organelle, nucleus in your case, first and isolate the 
protein as a second step. 

Immunoprecipitation can also be used to concentrate rare proteins from 
large volumes of tissue homogenate. A little trick here: Use dead Staph. 
aureus cells ("Pansorbin") to collect the immuncomplexes. The pelleted 
bacteria can then be boiled in sample buffer for electrophoresis.