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 
> only.  
> 
> 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

Good luck!



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