gel retardation assay method?
Luis M. Alvarez
luism at helix.nih.gov
Mon Feb 27 10:44:04 EST 1995
On 23 Feb 1995, Doug-young Ryu wrote:
> i am trying to perform gel retardation assay (mobility shift assay) with
> liver tisse nuclear extracts. It has so far given non specific bands
> If somebody has good protocol for this technique, please give me some
> good advice. (from nuclear extraction from liver to running PAGE)
> thanks in advance.
> dyryu at unity.ncsu.edu
I've been doing gel shift assays for a while witn nuclear extracts from
different tissues (including rat liver). The success in this technique
depends in the quality of ne nuclear extract, and what kind of oligo
probe are you using, this is what are you looking for? If you produce
nuclear extracts from tissues, you better make sure that they are good
quality ones. I suggest the employment of protease inhibitors, work in
the cold room at high speed.
To avoid, unspecific cross-reactivity I employ 1 ug of poly
[(dI-dC)(dI-dC)] per reaction. This compound is an artificial DNA that
will bind most of unspecific DNA binding proteins, but watch out! it will
also bind TBPs. If you want to see TBPs the best is to use poly
[(dG-cC)(dG-dC)]. Both polynucleotides are offered by Pharmacia.
I hope this will help you. However for a more detailed protocol you can
locate me at luism at helix.nih.gov or one of my partners
elopez at lambda.gene.cinvestav.mx
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