So I am trying to crystallize a Protein:DNA complex with DNA at at least 5 times excess at basic pH with a certain poleythene glycol molecular weight (PEG) and a mixture of soluble salts at low concentration less than 110 mM. I was getting a microcrystalline shower at a certain concentration of the components but lots of amorphous brown precipitate.
So I tried glycerol concentrations logically from 0 to 5%. I blocked the microcrystalline shower at glycerol concentrations greater than 1%. I kept increasing the concentration of glycerol regardless and at glycerol concentrations higher than 5% (v/v) and all other components in the system no brown amorphous precipitate appears; total protein is soluble. At glycerol concentrations greater than or equal to 15% (v/v) all other components in the system, the brown amorphous precipitate comes back. When glycerol is @ 13% (v/v) I got some small crystals not a showers worth. But nucleation seemed to fall asleep and that was it.
Then I lowered the concentration of one of the salts potassium citrate monohydrate to 100 mM and I get a reproducible white haze or white sheen appear over the droplet from the edge. The haze now seems to appear at any concentration of PEG much less than or equal to the concentration of PEG that I would expect to give me crystals. I also think the haze is growing.
I have seen this haze before when I crystallized the free protein under totally different conditions with different components in the system and acidic pH. This white haze would move from the edge of the droplet towards the center and then medium sized to large crystals would appear out of the haze in less than 30 minutes. Those crystals diffracted and a protein structure was solved. Of course under these conditions, crystals haven't grown out of the haze yet. Crystallizing the more conformationally inflexible tight binding DNA complex is more difficult than it logically should be.
Is there a term for this white haze or any information I can read about it?
What does it suggest to you?
Thanks!
To me, this looks like amorphous precipitate of your protein and maybe a sort of "crust" or skin of mother liquor that covers the droplet during vapour diffusion. I'm not sure what you are looking for, but this specific condition does not contain any crystals, and the precipitate does not really look pre-cristalline.
How did you come up with this condition? Have you tried using commercial screens to determine a suitable crystallisation condition before optimising this specific buffer combination? You usually don't optimise until you actually have a hit condition.
To me, this looks like amorphous precipitate of your protein and maybe a sort of "crust" or skin of mother liquor that covers the droplet during vapour diffusion. I'm not sure what you are looking for, but this specific condition does not contain any crystals, and the precipitate does not really look pre-cristalline.
How did you come up with this condition? Have you tried using commercial screens to determine a suitable crystallisation condition before optimising this specific buffer combination? You usually don't optimise until you actually have a hit condition.
This looks to me as Cloudiness which might certainly consist of either of the two macromolecules, DNA or protein or a combination of DNA protein complex. I would first study it by subjecting to agarose gel for confirmation of DNA and subsequently to SDS PAGE for protein analysis.
Adron Ung,
The white haze on the surface could be just the same precipitate you see deep in the drop, it might just look different depending on the reagents you have in the drop and the settings of your optics.
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