Hi Yang.
Adding to what Phoebe has said below, remember that when running polyacrylamide
shift gels using TBE and in the absence of SDS, your protein (depending on its
charge) may not migrate into the gel, or may in fact migrate out of the well
altogether (in the absence of DNA binding).
In order to monitor this a useful trick I devised is to load the well dry of
running buffer, and top it off carefully by floating a strong concentration of
acrylamide mix (ie a higher %age than the main gel) on top of the sample (your
loading buffer should keep the sample at the bottom of the well). Allow that to
set to form a plug, so that your sample is now trapped between the main gel and
this acrylamide plug.
Now pour in your well buffer and run the gel as usual. Afterwards EtBr stain
the gel, view / photograph under UV, then coomassie stain the same gel and view
/ photograph. That way you will have a record of both DNA and protein
movements, and you will see not only the protein-DNA complex migrating into the
gel, but also any non-migrating or opposite migration of the protein alone (it
should be trapped by the higher %age acrylamide in the plug). This allows you
to make a better judgement of the protein-DNA ratio required.
Acrylamide is better for this sort of job, but if you are running agarose gels
you can achieve a similar effect by placing your well former in mid-casting
tray when you set your gel. That way migration in either direction can be
spotted.
Hope that's useful,
John.
john.hi...@syngenta.com
www.syngenta.com
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phoebe
Rice
Sent: 01 September 2010 23:04
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to optimize protein-DNA complex conditions?
Dear Yang,
You might try acrylamide instead of agarose, since the caging effect of the
tighter matrix might keep the complex together better.
I'm not sure why MgATP? If your protein only binds in the presence of ATP you
might need a non-hydrolyzable analog?
Your concentrations are also vastly higher than those usually used for gel
shift assays. You might think that would gaurentee binding, but many DNA
binding proteins need quite a bit of salt to be soluble at high concentrations,
so your protein might all be stuck in the well. Actually, you can coomassie
stain an agarose gel with that much protein on it - that should tell you
exactly where the protein went.
Good luck!
Phoebe
Original message
>Date: Wed, 1 Sep 2010 14:55:07 -0700
>From: CCP4 bulletin board (on behalf of yang li
>)
>Subject: [ccp4bb] How to optimize protein-DNA complex conditions?
>To: CCP4BB@JISCMAIL.AC.UK
>
> Dear All,
> Recently I am working on a protein-DNA
> complex, and from running agarose gel, there is a
> weak delayed band after the band of pure DNA which
> indicates some DNA has bind to the protein though
> the binding efficiency is low. Then I tried to
> optimize the condition to increase the binding, but
>it
> did not work since the intensity of the delayed band
> didnot grow. The condition I used is list below:
> 1. the concentration of protein is about 1.5mg/ml,
> buffer in Tris-Cl, PH 8 and NaCl.
> 2. the running buffer for agarose gel is 0.5x TBE,
> PH 8.3.
> 3. different ratio of protein: DNA has tried, from
> 2:1 to 1:2.
> 4. different concentration of NaCl, MgCl2 and ATP in
> reaction system have been added, but no significant
> change.
> I wonder if there is any way to increase the binding
> efficiency? Is it possible to set up crystal plates
> in this situation, with protein and complex
> together?
> Any suggestion would be appreciated!
> Best
> Yang
=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp
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