sundares at bcm.tmc.edu (Sundaresan Venkatachalam) wrote:
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.