Hello, Nasir,
What concentration of your ammonium sulphate stock solution? Do your
solution remains immiscible without ammonium sulphate?May be addition of
concentrated solution of ammonium sulphate to PEG MME could cause phase
separation? Anyway, try to heat your solution a bit as recommended by
Tim or use ammonium sulphate stock solution with low ( probably 1 to 2
M) concetration and tell us if it works
06.12.2013 20:39, Tim Gruene пишет:
Dear Nazia Nasir,
did you warm up the solution a little? This makes working with PEG
solutions a lot easier. You may also note the Hampton do not maintain
the pH: they use the buffers as indicated but this does not mean that
the final solution still has pH 6.5. With your ingredients it probably
is, but e.g. with high concentrations of imidazole the final pH would
differ a lot.
Best,
Tim
On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote:
I have obtained crystals in Hampton Crustal Screen 2, condition 26.The
condition has the following components:
1. 0.2 M ammonium sulphate
2. 0.1 M MES buffer pH 6.5
3. 30% PEG mononmethyl ether 5,000.
When I try to prepare the screen in-house, I use the Hampton stocks of 3.5
M ammonium sulphate and 50% PEG mononmethyl ether 5,000. I make the MES
buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH
accurately. The water I use is filtered MilliQ. However, the PEG MME and
the rest of the solution remains immiscible and thus, forms droplets of
separated PEG when I set up crystallization. The original condition from
Hampton has no such problems and gives good crystals.
Can anyone let me know how to overcome the problem insolubility of PEG MME
in the buffer mix? I have not faced such problems with other conditions
which I prepare in house and have PEG in them. Further, it's not possible
for me to use the original screen all the time.
Thanks a lot!
Regards
--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: [email protected]
