gel retardation assay method?

Heather King hking at utxvms.cc.utexas.edu
Sun Feb 26 23:47:12 EST 1995


In article <3igqvk$hnd at taco.cc.ncsu.edu>, dyryu at unity.ncsu.edu (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)
> 
I've been doing gel retardation assays for the past few months and it took
me a while to get it optimized for my particular protein/DNA. Have you
tried varying the amount of non-specific competitor DNA (dIdC) that you
use in order to reduce non-specific bands?  
I'm working with thymus nuclear extract and in general this is how I set
up my assay:

Prepare 4% PAGE:
8 ml 5x TEB
5.3 ml 30% Acrylamide
26.4 ml dH20
Swirl, then add 250 ul 10% APS and 50 ul TEMED, pour
Let polymerize about an hour, pre-run gel for about 45 min. at 150v
(Use TEB as running Buffer)
While gel is pre-running I set up my sample reactions.  If it's the first
time I've tested a particular batch of extract I set things up as follows:
(in mfuge tubes)
Tube#          1     2     3     4     5     6     7     8    

dIdC           1ul - - - - - - - - - - - - - - - - - - - > 
(1ug/ul)

Buffer*        10ul - - - - - - - - - - - - - - - - - - ->

nuc.extract     0   0.5ul  1ul   2ul   3ul   4ul   5ul   10ul
(~2ug/ul)

I mix the dIdC, Buffer(*whatever buffer your nuc. extract was prepared in;
I use 20mM HEPES, 0.1M KCl, 20% glycerol + 0.5mM PMSF,1mM DTT and loading
dye added at last minute), and nuclear extract. Incubate 25C for 5 min.
Then add 1 ul of 32P labeled DNA probe (~0.2ng/ul)
Incubate 25C for 20 min.
Load onto gel and run at 150v for 3 hrs (that's for a 200bp DNA probe)
Dry and place on film overnight.

I hope this can help you. Let me know if you have any more specific questions.

Heather King
Grad Student, Microbiology
UT Austin
hking at utxvms.cc.utexas.edu



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