2 replies to this topic

[anonymous]

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

[diliana]

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!

[aimikins]

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!