jm6 at st-andrews.ac.uk
Wed Apr 15 07:01:55 EST 1998
On 14 Apr 1998, Elsbeth Walker wrote:
> Could anyone tell me a typical concentration for the DNA probe used in a
> bandshift assay? I'd hate to end up wasting expensive oligo if I don't
> have to!
First of all, you should radiolabel some double stranded oligo. 5 pmoles
of your oligo is probably the best. I do not know what method for
radiolabelling you use (you did not tell us anything about it, if you want
more info tell me more about how you label it).
Then, you have to find a way to separate your ds oligo from the
unincorporated mononucleotide(s) (electrophoretic separation in a TBE
polyacrylamide gel, size exlusion chromatography etc).
The final volume of your ds oligo (in TEN -also called STE) should not
exceed 200ul. The concentration of the oligo should be about 20-25nM.
The only way you can see how much of it you need to add is by adding
increasing amounts of your oligo to a certain amount of your protein
extract. Then, you decide the volume of your oligo you need to add, by
developing a film. You will choose the volume of your oligo that you see a
discent band. That volume depends on the protein amount and on the
affinity of your protein for your oligo.
> Elsbeth L. Walker
Hope that helps,
PS If you want more, please tell us more about what you want to do.
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