problems with Band Shift Assay

micjms at lure.latrobe.edu.au micjms at lure.latrobe.edu.au
Sat Apr 5 00:48:09 EST 1997


I have been having problems with DNA Mobility Shift Assays. The problem doesn't
appear to be the DNA binding reactions as such. DNA fragments were isolated
ranging from 140 - 400 bp and labelled them with 32P. The DNA binding protein
was purified using GST fusion. Binding reactions were performed with and
without the protein ie. control DNA binding reaction and DNA & protein binding
reaction. Samples were run using the Phast System; 8-25% gradient gels and
native buffer strips. The problem involves the DNA not running into the gel,
this occurs also in the absence of the protein. Due to the DNA being GC-rich I
tried adding DMSO prior to loading the samples on the gel, however this did not
make any difference. 

If anyone has experienced the same problem or has any hints please drop me a
line. 

Thanks

Joanne


Joanne M. Santini
School of Microbiology
La Trobe University
Bundoora, 3083
Melbourne VIC
Australia
Ph: +61 3 9479 1305
Fax: +61 3 9479 1222
E-mail: micjms at lure.latrobe.edu.au 



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