Problem in purification of a bacterial transcription factor - Help...please....I only have 5 months left (Nov/06/2005 )

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, then ammonium 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!

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I'm sure there are people here that will be more help than I can be with column chromatography, but your post reminds me of a problem I had once with a less ambitious protein purification -- I was able to detect the protein early, but not after storage. It was driving me nuts until I found the answer -- the protien was sticking to the tubes I was storing my fractions in (they were polystyrene, as I recall).

Any chance this is what's going on?

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I think this is not the case, since I have stored the protein in both glass tubes or polypropene tubes and these seem not the culprit. Anyway, thanks!

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when you take the flow-through fraction, perhaps the wash buffer or whatever the protein is eluting with, is at a pH that results in an unstable conformation

have you tried any alternate elution buffers?

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The tablets are a good protease inhibitor start, but I'd also try PMSF (or more safely, Pefabloc).

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