I am curious to know if there are any good non-isotopic methods for labeling oligos to be used for DNA-protein interactions in mobility shift assays

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Hi,
You can try the DIG-labelling kits from Roche or directly label your target DNA by PCR reaction with 5'-DIG labelled primer. It is easy and gives good results.
For more information see the site of Roche (Boehringer Mannheim).

I have tried it and find it very useful.

Good luck!

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It is also possible to biotinylate your DNA oligo probe for use in EMSA. It is important to end-label your probe (internal labeling can hinder the protein-DNA interaction). I have had much success with a DNA biotinylation kit from Pierce. The only drawback is that you have to label your oligo strands separately, then anneal them; the labeling reaction doesn't work well for dsDNA.

Then, it is only necessary to perform your favorite method for the binding reaction, run the gel, perform a transfer, and detect the DNA using a streptavidin system (I have had good results detection non-radioactive DNA with kits and/or reagents from both Pierce and NEB). I use non-radioactive EMSA and I can go from labeled probe and nuclear extract to photographing the results in well under 24 hours.

Good luck!

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Hi,

we also have a problem with EMSA...

We use endlabelled biotinylated dsDNA (labelled with Pierce-Kit) an want to do EMSA with the Pierce lightShift-Kit...

There are no problem with the gel, our "simple" problem is to DETECT the biotin-dsOligos (some 30mers) on the gel, we can detect the non-labeled DNA but not the biotin-tagged version. The results are the same with the positive control of Pierce?

We can´t move on without solving the problem!!

Do you have a helpful hint??

Thank you very much in advance!

Best wishes,

Gerhard

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