How to show that the proteins are really labeled?

Dr E. Buxbaum EB15 at le.ac.uk
Fri Aug 1 11:20:47 EST 1997


calvert at eos.ncsu.edu (Phil Calvert) wrote:
>In article <5rnko6$qm8 at falcon.le.ac.uk>, "Dr E. Buxbaum" <EB15 at le.ac.uk> wrote:
>
>> The phenol can be removed by extraction with diethyl ether (2 times twice 
>> the volume of your sample). This will leave proteins in the small amount 
>> of water which was dissolved in the phenol.
>
>Thanks for the reply.  Yes, I am aware of that method.  I even used it once
>about a year ago.  However, I don't really trust it.  When I used the
>method I got a lot of fluffy precipitate which wasn't expected (the paper
>first describing the method doesn't mention it).  Although I don't have
>proof, I suspect that the diethyl ether may be denaturing the protein. 
>Maybe not all the protein, but it is plausible that some of the protein
>could be denatured by this procedure.

Phenol is an acid as well as an organic solvent. One would therefore expect 
the proteins to be denatured. The question is wether the SDS-sample buffer 
will bring the precipitate into solution again (addition of urea might help) 
to allow PAGE. This can be found out only by trial and error. I know of at 
least one protein (Mdr1), that can not be redissolved after phenol 
extraction. 

On the other hand, SDS-PAGE is a simple enough method that the experiment is 
worthwhile. If it does not work, I can think of only one alternative approach. 
Remove the phenol as above, precipitate the proteins completely with TCA 
(measure the radioactivity in the supernatant) and hydrolyse the putative 
proteins in HCl (6 n, in vacuo, 110 degrees over night). Separate the amino 
acids by 2-d thin layer chromatography (or by HPLC, if you have the equipment) 
and make an autoradiogram from the plate. This should give you radioactive 
spots in the position of those and only those amino acids, which can be formed 
metabolically from the radioactive precursors you fed your bugs. 




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