Thanks to all for the suggestions! We will try, this weekend at macCHESS,
all those conditions for which we have reagents, and order the others now.
> On Oct 16, 2012, at 9:01 PM, "Edward A. Berry"<[email protected]> wrote:
>
>> Would someone suggest a cryoprotectant for index screen #59? It contains
>> 0.02 M MgCl2
>> 0.1 M HEPES pH 7.5
>> 22% polyacrylic acid 5100, Na salt
>>
>> Adding glycerol tends to dissolve the crystals.
>>
>> Thanks,
>> Ed
>
Yvonne TAN Yih Wan (ETC) wrote:
I had a similar condition. I have used increased concentration of polyacrylic
acid 5100 as cryo. Gradual increase of 10%glycerol + 10% E.glycol in mother
liqour can aslo be used as cryo, in my case.
Roger Rowlett wrote:
Glucose, 15-25% depending on how good polyacrylic acid is as a cryoptectant
itself, is worth a try. It doesn't
usually dissolve proteins as readily as glycerol. Ethylene glycol is also an
option.
[email protected] wrote:
I obtain crystals in the same condition. Obviously the cryo that works for me
may not work for other proteins, but I use 10% ethylene glycol and soak the
crystals anywhere from 10 minutes to one hour. The other thing that seems to
make a huge difference for me is the way in which I cool the crystals.
Sometimes freezing in liquid nitrogen versus freezing directly in the
cryostream can be the difference between 5 angstrom high mosaicicity data and 2
angstrom high quality data. I have obtained good data from both freezing
processes.
Hope this helps, it would be interesting to know how it turns out for you.
*note: the above methods are for a membrane protein. May or may not make a
difference for soluble proteins.
Francis E Reyes wrote:
Oil?
David Cobessi wrote:
Dear Ed,
Have you tried paratone or another oil?
Best regards,
David
Benz, Joerg wrote:
did you try paraffin oil already (dry oil, can be found i.e. in the Cryokit
from Hampton). Worked for me in nearly all difficult cases. You just need to
make sure that all of the surrounding solution around the crystal is replaced
by the oil.
Ulrike Demmer wrote:
try ca. 30 % PEG 400
Another possibility could be Perfluoropolyether Cryo Oil from Hampton Research.
This liquid doesn't mix with water. You put the crystal in few microliter and
remove the excess water droplets from your screen solution (otherwise you will
get ice rings).
Anthony Savill wrote:
Please have a look at our new product CryoProtX.
http://www.moleculardimensions.com/shopexd.asp?id=3732
Enrico Stura wrote:
Glycerol is a solubilizing agent. Unless something is done to conteract such
effect
the crystals will dissolve. Laura Vera from my lab made a presentation at
ICCBM-14 on this subject.
We have developed a set of balanced solutions, mixed solubilizers like glycerol
and ethylene
glycol with precipitants like MPD, DMSO and neutral compounds like di-ethylene
glycol
and propanediol that with PEG and salts crystallization conditions take
out the guesswork from designing cryo-conditions.
The mixed conpounds are marketed by Molecular Dimensions google search:
CryoProtX
Before ordering the kit check that the crystals do not dissolve with MPD. If
crystals crack
but do not dissolve with MPD (it may be worth ordering the kit). Cracking is
good since it means
that the crystals do indeed dissolve in glycerol because of glycerol's
solubilizing properties.
Now try Propylene glycol (1,2-propanediol).
This is a neutral single component. It is a good starting point to work on
your cryo-conditions which you will then be able to improve when you have all
the various
cryo-components and premixed combinations.
The paper for the mixed cryosolutions will be submitted to the ICCBM-14
special issue of the Crystal Growth and Design probably this week.
polyacrylic acid is a novell precipitant, make sure that you keep
magnesium in your cryosolution. I would also check if increasing the magnesium
concentration
in the cryoprotectant solution might not be beneficial.
On Oct 16, 2012, at 9:01 PM, "Edward A. Berry"<[email protected]> wrote:
Would someone suggest a cryoprotectant for index screen #59? It contains
0.02 M MgCl2
0.1 M HEPES pH 7.5
22% polyacrylic acid 5100, Na salt
Adding glycerol tends to dissolve the crystals.
Thanks,
Ed
