gel shift assay

William K. Pierce natldiagnos at mindspring.com
Mon Mar 17 09:16:15 EST 1997


Joachim Kremerskothen wrote:


> we have some problems with our gel shift assays because our protein/RNA
> complex do not run on a native 5% polyacrylamide gel using TBE buffer (pH
> 7,6). Any suggestions for another gels system, etc.?

My feeling is that the gel and buffer system can be awfully
protein-specific.  
Back in the days when I was doing gel-shift, I got the best results for
my 
protein (the dioxin receptor) using 20mM Hepes-20 mM Tris-1 mM EDTA-6%
PA 
(19:1 acrylamide:bis-acrylamide) gels (pH 8).

AJW
----------------
John Watson
Bristol-Myers Squibb Co.
watson_j at bms.com


ANOTHER LIKELY POSSIBILITY IS THAT THE PROTEIN/RNA COMPLEX IS "NOT
RUNNING" ON THE 5% GELS BECAUSE IT IS FORMING SUCH A HIGH MOLECULAR
WEIGHT COMPLEX THAT IT CANNOT ENTER THE GEL.

A QUICK WAY TO CHECK THIS WOULD BE TO CAST A 1.5% STANDARD AGAROSE,
HORIZONTAL AGAROSE GEL IN TBE. LOAD YOUR PROTEIN/RNA COMPLEX AND SEE IF
IT MIGRATES. IT WILL WORK TO DO THE ELECTROPHORESIS AS A SUBMARINE GEL. 
SEE THE FOLLOWING PAPER FOR RECOMMENDATIONS ON HOW TO CAST AND RUN THE
GEL:

AGAROSE GEL ELECTROPHORESIS OF HIGH MOLECULAR MASS PROTEIN COMPLEXES
J.R. THORNTON, H.A. DAUM, III AND S.T. CASE
BIOTECHNIQUES
VOL. 18
NO. 2
1995 PAGES 324-327.


HANK DAUM
NATIONAL DIAGNOSTICS
EMAIL: natldiagnos at mindspring.com



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