Problems in purifying a bacterial transcription factor

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Problems in purifying a bacterial transcription factor

Post by janel » Mon Nov 07, 2005 5:14 am

I am trying to purify a novel transcription factor from a bacteria. The only thing I know is the DNA sequence that the protein binds.

What I've done is protamine sulfate precipitation and anion exchange chromatography (in which the protein appears in the flow-through fraction).

Up till this step, problem arises! I can detect the presence of the transcription factor by band-shift assay just after I run the protein through the anion exchange column. However, when I stored the active fraction overnight (either in 4oC or -20oC), the binding activity lost in the next morning.

I have though of some possible sources of this problem (please correct me if I am wrong):
1) protease --> I have tried adding protease inhibitor cocktail tablet, but it doesn't help
2) loss of co-factor --> if this is the culprit, then I shouldn't be able to do the band-shift just after the chromatogaphy step, right?
3) precipitation --> I didn't get any pellet by centrifugation of the active fraction

And I have tried to add glycerol to the protein, but I found that glycerol will affect the band-shift assay and the latter chromatographic steps.

Please give me some advice on preserving the protein!

Also, I have tried rushing to do several purification steps within a day (before the activity lost). The next step after anion exchange chromatography I chose is DNA-affinity chromatography (by means of Dynabeads). However, I found the protein band on SDS-PAGE very faint. I afraid I can't do any amino acid sequencing or MS-MS with this faint silver-stained band.

Can anybody help me and give me some suggestion? Thanks a lot!

King Cobra
King Cobra
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Post by sdekivit » Tue Nov 08, 2005 5:25 pm

is the pH of the solution your protein is in at the right value ? maybe you protein denatured due to a wrong pH

another explanation could be that the protein that binds to the DNA is part of a complex. If this is so, immunoprecipitation could be an option.

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