How to show that the proteins are really labeled?
Dr E. Buxbaum
EB15 at le.ac.uk
Wed Jul 30 09:57:10 EST 1997
calvert at eos.ncsu.edu (Phil Calvert) wrote:
>Please excuse me if these sound like stupid questions; I don't normally do
>this kind of work.
>We have fed a radiolabeled polymer to some bacteria and then did an
>extraction/fractionation on the culture. One of the fractions is a phenol
>solution that supposedly contains proteins.
>What we want to know is whether the bacteria incorporated any of the
>radiolabel into their proteins. The extract contains 5564 dpm/ml of C-14,
>so there is a good chance that there is labeled protein in there. However,
>how can we be sure that the counts are coming from protein and not from
>something else? Is there a simple procedure for getting pure protein out
>of the phenol extract? We don't care which proteins are labeled so,
>obviously, anything that separates the proteins from each other would be
>overkill. Any ideas?
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. The next step normally would
be SDS-PAGE to separate proteins by MW and autoradiography (or
fluorography after soaking the gels in PPO). This may give you more info
than you need, but it is a simple and straight forward method. If
however you do decide that you need even more info, blotting the proteins
onto PVDF and sequencing would provide it.
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