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A few additional ideas:
- The typical (and convenient) set up is to equilibrate a mixture of protein and precipitant solutions against the very same precipitant used to mix with the protein (“well solution”). Try either changing the precipitant : protein volume ratio to a larger protein volume than well solution (you will need to re-screen precipitant concentration). Or try equilibrating against a well solution which contains more precipitant than the solution which is added to the protein (you need to set up two sets of solutions, one to be mixed with protein and one to be dispensed to the well – the concentration difference between well solution and solution mixed to the protein needs to be screened).
- Also beware of temperature. Temperature fluctuations may lead to crystal dissolving. The fluctuations may occur even if you keep the trail in an incubator because you need to move the trail to the room where you observe the crystals. I’ve had this issue last year. In my case: the crystals were dissolving after transfer from a 18°C to RT room and put back into the 18°C. The solution in that particular instance was to grow and store the trail in a 22°C incubator. Details, details J
Good luck
Thierry
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of NISHANT SINGH
Sent: Friday, September 02, 2016 9:28 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Crystal Growth
Thanks for all the advice so far.
I have the protein in 10mM MES and 20 mM NaCl at pH 7.5. There is already heavy precipitate in the background, hence it is difficult to comment on if the crystal disintegrates into precipitate or not.
On Fri, Sep 2, 2016 at 9:00 AM, Patrick Shaw Stewart <[log in to unmask]> wrote:
Two comments Nishant
(1) You don't have much salt in your reservoir solution. How much salt have you got in your protein buffer? If more than 200 mM, your vapor diffusion experiment may be going backwards - water may be going from the reservoir to the drop, which is slowly becoming more dilute. Do the drops seem to be growing?
(2) (Best answer) use your crystals to make a seed stock and run a "random" microseeding screen : )
I hope it helps you
Patrick
On 2 September 2016 at 12:49, NISHANT SINGH <[log in to unmask]> wrote:
Hi All,
I have been working a 45kDa protein that I am able to crystallize at room temperature in 9% PEG8000 and 200mM Ammonium Sulfate at 4-6mg/ml. I see plate like crystals appear within 24-48 hours of setting up the tray, along with heavy precipitation in the crystal drop. However, most curiously the crystals seem to disappear as time progresses. Any idea why that might be the case and what can I do to prevent it other than harvesting the crystals within 24-48 hr time period?
Sincerely,
Nishant
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