DNA pull down strategy to purify a transcriptional regulator

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adlheras
adlheras's picture
DNA pull down strategy to purify a transcriptional regulator

Hi everybody

I want to capture a bacterial transcriptional regulator by using Dynabeads®M-280 Streptavidin coupled to the dsDNA cognate promoter. I followed the manufacturer’s instruction to Immobilize the biotin-labelled dsDNA onto the beads . I got 5 micrograms of dsDNA attached to the beads. By using these beads i tried to purify the transcriptional regulator using different KCl concentration in the buffer (50, 100 and 150mM). I recovered 18% of the protein from bacterial extracts using 100mM that I could detect by western blot. When I run a Coomasie i saw other proteins that were eluted from the beads, so I could not get a proper purification of the protein. My question is: is there any method to optimize the transcriptional regulator recovery in terms of amount and purity

Thank you very much

Chin Fen Teo
Chin Fen Teo's picture
Hi adlheras,

Hi adlheras,

Two questions;
(1) Is the transcription factor of your interested shown as the major component on the coomassie stained gel regardless the presence of other proteins?
(2) Can you please provide the full recipe of your binding/washing condition?

Cheers.

adlheras
adlheras's picture
Thank you very much for your

Thank you very much for your help!!.

It is not the major component in the coomassie gel , I only can detect it by westernblot .

the binding/washing buffer that I used is this:

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10mM Tris-HCl pH 8.0
10 mM MgCl2
5mM Ca Cl2
1mM EDTA
0.2 mM DTT
10% glycerol
100mM KCL

I need to get the transcription factor as much as pure as this thecnology allows. D you need if it is possible to otain a pure protein by using this DNA pull down ??

Thank you again for your kindly help

Chin Fen Teo
Chin Fen Teo's picture
Hi adlheras,

Hi adlheras,

Threorically, I don't see any problem using this approach to enrich for your transcription factor of interested. There are a few conditions you can optimize-

(1) You can preclear your sample with Streptavidin beads alone before the pull down experiment to eliminate any potential non-specific binding.

(2) You might want to adjust the ratio of your sample and dsDNA-conjugated beads- too much of the dsDNA-conjugated beads will lead to non-specific binding too.

(3) You can consider adding non-ionic detergent (such as NP40) in your wash buffer.

Good luck.

adlheras
adlheras's picture
Thank you very much !!!!

Thank you very much !!!!

I wil try your suggestions

Best