gel retardation assay

Loïck Le Dantec thesax at
Thu Jun 10 12:18:47 EST 1999

On Thu, 10 Jun 1999 16:44:30 +0200, Inge Depoortere
<inge.depoortere at> wrote:



>I want to set up a gel retardation assay for NF-kappa-B. Can someone
>give me a good and quick protocol for the preparation of the
>radiolabeled oligonucleotide probe. I will start from a synthetic single
>stranded probe (20bp) and want to have a double stranded labeled probe.
>Can I use the end labeling procedure with T4 polynucleotide kinase?  How
>should I make it double stranded. First label and then anneal (under
>which conditions) or first anneal (how to do) with an overhang and then
>label .
>Thank you

Here's the protocol i used for gel retardation with a 30 bp double
stranded probe:
100 pmol single stranded oligo incubated with 10u T4 P.K. and 10 µl
[gamma32ATP](3000Ci/mmole), 1h, 37°C. Purification on sephadex G-25
column and annealing (in TE buffer, heating and slowly cooling to
avoid self annealing of the inverted repeat sequence...not sure it is
really useful).

(sorry for my english)

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