A new transcription factor

mbarnhart at my-dejanews.com mbarnhart at my-dejanews.com
Wed Sep 2 11:11:42 EST 1998


Yoshi,

Your approach depends upon your goals.  I can think of three possibilities
offhand.

First, you can transfect the factor into cells and do differential display
PCR. DDPCR is likely to identify genes that are up- or downregulated,
directly or indirectly, by this factor.

A second possibility is to do a procedure that I've heard of referred to as
SELEX.	In this procedure, you bind your protein to a mix of DNA's with
defined ends but random sequence in the middle.  You precipitate your protein
with the bound DNAs (via GST fusion or similalr procedure) and perform PCR
with primers to the defined ends.  You do about three rounds of this
selection and then clone and sequence the selected DNAs.  This will help
define the binding site of your protein.

Third, you could generate antibodies to your protein and do
immunoprecipitations to see what proteins interact with your new protein.  I
would suggest doing the precipitations on cytoplasmic and nuclear fractions
separately.

By the way, the SELEX will only work if your protein binds as a monomer or a
homomultimer. If it needs a different protein for mulitmerization, then you
probably won't get sufficient binding for selection... and anything you do
select may not mean anything.  It's still worth doing, but keep this in mind.

Michael Barnhart
Connective Tissue Physiology Lab
University of Houston


In article <v01540b00b2126cc85b64@[130.194.152.53]>,
  yoshi at COBRA.PATH.MONASH.EDU.AU (Yoshito Tsukada) wrote:
>
>
> Dear netters,
>
> I have recently cloned a putative trancription factor. Because it has
> Küppel type  zinc finger, PHD finger, and four clusters of proline rich
> domains, it is highly poosible that it forms trancriptional complex. But
> this clone was found by accident without any background, I do not know how
> to examine the function of this protein. Does anyone know how to start
> studies about the interaction of new factor and other DNA motifs or
> proteins?
>
> Yoshito Tsukada
> Monash Uni, Australia
>
>

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