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
<christine.har...@fda.hhs.gov> 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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Enrico Stura
Sent: Monday, November 28, 2011 1:45 PM
To: CCP4BB@JISCMAIL.AC.UK
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 <yj...@brandeis.edu> 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: est...@cea.fr Fax: 33 (0)1 69 08 90 71
