gel retardation assay

Loïck Le Dantec thesax at club-internet.fr
Thu Jun 10 12:18:47 EST 1999


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

>Hi,

Hello,

>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
>Inge
>

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).

Loïck
hth
(sorry for my english)



More information about the Methods mailing list