5 replies to this topic

[Ambrósio]

Hi everyone!

Does anyone know if it is possible to do a gel shift (DNA-protein interaction) with a 35S-labeled protein instead of the 32P-labeled DNA?

I've been told that it is possible, but have failed to find any protocol...

I'm producing my protein in vitro (TNT promega kit).


Thanks in advance!

Edited by Ambrósio, 27 July 2009 - 06:42 PM.

[bob1]

It should be, just label and extract your protein then run the EMSA as normal.

[mikew]

The TNT kit comes with a protocol for S35 labeling.
However, the signal is weaker than 32P labeling of your DNA probe.

[Ambrósio]

I was wondering if I just needed to add the DNA to the translated protein, incubate for a while and then run a non-denaturing gel and check if the protein shifts. It sounds too easy...

[mikew]

The important thing (VERY IMPT!)
is to do a negative control with rabbit lysate
that does not have your protein. Eg. 35S label another unrelated protein as a negative control.
The lysate will bind many many probes.
If you do not have a negative control, label the oligos with 32P and
use lysate as a negative and your translated protein as the unknown.
   There are many many proteins in rabbit reticulocyte lysate that bind DNA,
it is very easy to get artifacts.

[Ambrósio]

Cool. Thanks a lot!