Dear Christine,
I had guessed that you had more salt in the protein solution
than in the reservoir with a relatively low protein concentration.
The protein is in:
> 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration
> of ~4.3mg/mL.
and the reservoir is:
>>> (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and
>>> 30% PEG MME 550).
We can suspect that no matter what temperature the equilibrium will move
towards dilution in the drop.
Basic suggestion:
Set up a range from 20-30% PEG MME 550
with at least 150mM NaCl in the reservoir.
Fine tuning:
Add high MW PEG (10K or 20K 1-5%) it should have a stabilizing effect
Try a range of salts apart from NaCl: The clasics are: Li2SO4, KSCN, MgCl2.
Try additive amounts od MPD 0.5-3%.
Try a small tight range of various pH.
Enrico.
On Mon, 28 Nov 2011 21:43:51 +0100, Harman, Christine
<[log in to unmask]> wrote:
> Hi,
> Thank you so much for your replies. A lot of you have mentioned
> fluctuations in temp as the major contributor. And a few of you have
> asked for more details of my protein/buffer and set up. To my
> knowledge, the tray has been kept at constant 20 C (in an incubator)
> with exception of course to when I remove the tray to view the drops.
> It could be possible that my inspection of the tray might have
> contributed to an increase in temp, but only temporarily. I am very
> careful about the time the drop sees intense light, but it is possible
> the temp could have changed enough to cause this problem. Just to give
> a few more details. My protein (a Fab fragment/peptide complex
> (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with
> 150mM NaCl at a protein concentration of ~4.3mg/mL.
After setting up my
> drop with reservoir solution I add NaCl to well to give ~75mM NaCl to
> match ionic strength of protein in drop which is diluted 1:1 with well
> solution. I do hope this problem is temperature. Although I am a
> little sad to not be able to freeze those crystals I did see, I still
> consider myself lucky to get such good result from a condition right
> from the screen so there will be some definite optimization set ups with
> this condition. Could I safely say though that the crystals I observed
> are not salt..:) I guess that is one good thing to take from this. Any
> more suggestions on optimization would be very welcomed.
>
> Thanks again to all you,
>
> Christine
>
>
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Enrico Stura
> Sent: Monday, November 28, 2011 1:45 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Disappearing crystals
>
> When advice on crystallization is needed, it is important to give details
> of
> the protein concentration, the buffer the protein is in as well as the
> method
> used to grow the crystals.
>
> Problem: The crystallization conditions are essentially low salt: 100mM
> buffer
> and only 50mM CaCl2. So the buffer that the protein is in is very
> important !!
> Fluctuation in the reservoir/drop environment will lead to crystals
> dissolving.
>
> Solution: Balance the salt in the reservoir and in the
> protein:precipitant
> drop and make sure
> the temperature is kept constant.
>
> Since I do not have all the necessary information, the diagnosis and the
> solution proposed
> are likely to be wrong!
>
> Enrico.
>
> On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin <[log in to unmask]> wrote:
>
>> On 11/28/11 12:04 PM, Harman, Christine wrote:
>>> Hi All,
>>> I have just noticed a very strange thing and need some help in
>>> understanding it. I recently found two crystals in a condition from a
>>> screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and
>>> 30% PEG MME 550).
The small crystals appeared after a month and
>>> started to grow over the next 5 days after I first saw them (see
>>> pictures attached). I just check the same drop today and now the
>>> crystals are gone. So I was wondering what happened and if anyone
>>> experienced this before. Any insight or advice on what to do would be
>>> greatly appreciated.
>>> Thanks
>>> Christine
>>> Small 5 days later
>> Hi Christine,
>> I had similar experience. In my case, another crystal showed again with
>> different size a few days later. Sometimes, it seems like it is a common
>> event to others as well as I heard although my case only takes about a
>> week to be crystallized. I'd rather wait or just set up again or in a
>> slightly different way.
>>
>> Wish you well.
>>
>> Young-Jin
>>
>
>
--
Enrico A. Stura D.Phil. (Oxon) , Tel: 33 (0)1 69 08 4302 Office
Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab
LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE
http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura
http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
e-mail: [log in to unmask] Fax: 33 (0)1 69 08 90 71
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