Soaking in nucleic acids into a PAGE gel

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Fri Jul 13 06:04:10 EST 2001


In article <3B4DDB92.85E689AC at bones.biochem.ualberta.ca>, the eminent
Karl Fischer at GHRI wrote
>My colleague is concerned that the proteins of interest are showing
>anomalous migration patterns potentially due to interaction with the
>polynucleotide during the electrophoresis. By way of example, an enzyme
>with an amino terminus deletion shows a slower migration in the activity
>gel than the parent enzyme even though the deletant is only 2/3 as large.
>He was hoping that leaving out the template from the matrix followed by
>template soak-in would give a better sizing comparison.

Given that the polymerase is a DNA binding protein, that is not too
unexpected. I wonder if he can blot the protein out onto nitrocellulose
and incubate the nitrocellulose with the polynucleotide in solution.
It's a bit like Boehringer's as was patent for assaying recombinant
pols. By blotting E.coli colonies to nitrocellulose, heat lysing the
colonies on the filter and assaying using radiolabel. The template was
that present in the lysed colony. I tried their method a few years ago
after it was published in Gene and it worked great.

Duncan
-- 
It might look like I'm doing nothing, but at the cellular level I'm really
quite busy.

Dr. Duncan Clark
GeneSys Ltd.
Tel. +44 (0) 1252376288
FAX +44 (0) 8701640382




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