dk at no.email.thankstospam.net
Wed Jan 9 00:03:57 EST 2002
"user" <q6252397 at fernuni-hagen.de> wrote:
>I would like to purify a transcription factor with DNA-Affinity
>I bought a Streptavidin-Superflow Agarose resin. I'd like to couple
>biotinylated DNA-Fragments (80bp) on the column, to get active TF.
>The resin has a Biotin binding capacity of 350 nmol/ml resin.
>The problem is to get that much biotinylated DNA.
350 nmol of 80-mer = 80*300*350/10^6 mg ~ 8 mg. I'd try to
not use more than 50% of max capacity then. Still, you can't
synthesize that much. Here is what I'd do: clone that 80-mer
by cheap unique site like EcoRI into some plasmid (if binding
is to 80-mer then probably more than 80-mer would be a lot
better for chromatography). Such cloning is easy - mix strands
designed to make sticky ends at 10-100 uM, boil in ~ 300 ml beaker
(heat sink), let cool slowly at rt to ~ 37C, ligate with a series
of 10-fold dilutions. One of the dishes always gives lots of
clones. If you use one site and get a clone with concatameres -
even better (higher yeild of the product upon complete digestion).
Now you have an unlimited supply of cheap sequence of interest.
You can always label it with biotin - using "photobiotin" or by
enzymatic end labeling with analogs (the former will be a lot
cheaper but less clean as far as chromatography behavior goes).
The real problem arises if your factor only binds ss DNA. In this
case I'd forget about biotin and use good old coupling to BrCN-
activated sepharose in 90% formamide (to keep DNA denatured).
See, for example, Van Eekelen et al, 1981, Eur. J. Biochem,
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