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EMSA Problems, Mystery Band :(

EMSA

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#1 evilhamster

evilhamster

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Posted 03 June 2012 - 04:50 AM

Hi all,

I've been asked to run an EMSA (gel shift) assay on a DNA-binding protein towards a synthetic oligo. We have a very high concentration of the stock (50uM), making about 0.5uM of the oligo duplex. Given this, we dedcided to just use EtBr to stain the gel instead of using a radiolabelled probe

Single strands of the oligo were dissolved in 10mM HEPES, pH 7.5, 150mM NaCl, 1 mM DTT, combined at equimolar amounts, heated at 95C and allowed to cool to room temp. overnight to form the duplex.

Agarose gels (2%) were prepared with TAE buffer and EtBr was added to the molten mix before allowing to cool and harden.

The EMSA mix contains 0.5uM of the DNA duplex, and increasing amounts of protein (5-100uM) were found to create a band of increasing thickness which was 'heavier' than the free DNA.

When I ran a 'free protein' control in the gel though, a band was present with the same relative mobility as the band observed in the 'protein+DNA' lanes. Running the protein through a heparin column to remove what was originally thought of as a DNA contaminant was ineffective; the band was persistent. Mutants used in previous studies with the crystal structure resolved showed the same band.

I tried adding guanidinium HCl, to maybe 'denature' the protein, thinking that I'd see the 'free DNA' band migrate, but the whole complex didn't migrate.

This anomaly has only happened in 'my' gel Posted Image

What's that mystery band, please help Posted Image
(SEE ATTACHED)emsa problems.png

Help Please Posted Image

Edited by evilhamster, 03 June 2012 - 05:14 AM.






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