From: Browning Christopher
Date: 27 September 2011 16:55
----------
From: Mark J van Raaij
Hi Christopher,
you'll have to try to find out how they mix the components at MD and if they pH the solution afterwards or before (or not at all...) and to which pH. You can ask them; or try different mixing/pHing protocols on a small scale and see if one works in keeping all in solution.
Mark
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
----------
From: Enrico Stura
When you give the composition of the screen condition, you must also give the conditions of
the protein buffer, since you get crystals when the two are mixed.
If you analyse many small salt crystals by SDS-PAGE you may still get staining since protein
will precipitate on the salt crystals.
Enrico.
--
Enrico A. Stura D.Phil. (Oxon) ,
----------
From: Mark J van Raaij
PS I would also try substituting imidazole for HEPES and zinc sulphate for zinc chloride and sodium sulphate.
----------
From: Roger Rowlett
Imidazole is basic. If you mix it directly with zinc salts you will get zinc hydroxide. Most screens are prepared by mixing stock solutions. In your case I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50% PEG-4000. Add the PEG last. This should work. You may or may not need this specific salt or buffer to get crystals, and could swap them out or change concentration as required. 50 mM buffer may not be sufficient to control condition pH depending on the protein storage buffer composition.
Roger Rowlett
----------
From: Enrico Stura
The PEG could also be the problem, so you can mix your stock solutions by the method described
below and end up with the same result as soon as you add the PEG.
See:
Frances Jurnak: Effect of chemical impurities in polyethylene glycol on macromolecular crystallization
Journal of Crystal Growth
Volume 76, Issue 3, 2 August 1986, Pages 577-582
Enrico.
----------
From: Christopher Browning
Thanks for the replies. I think I got it figured out. The pH seems to be
the definitive factor. Indeed, the more alkaline the pH, the higher the
amount of precipitation. I just played a bit around with the pH of
Imidazole and it's cleared up. It also seemed to help to add things in
this order..... Imidazole--->water--->ZnSO4--->PEG.
Cheers,
Chris
Switzerland
Date: 27 September 2011 16:55
Dear All,
--
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
My question might be a bit out of place, but perhaps someone can help. I've screened my protein in the Nuc-Pro screen from Molecular Dimensions and found some crystals in condition B10. They seem to be protein crystals as they are not highly optically active and look different to salt crystals. So condition B10 consist of 10% PEG 4K, 50mM Imidazole pH 7.2, 20mM Zinc sulfate. In the screen, this condition is perfectly clear, but when I try and make my own screen, the whole solution turns white. Apparently, Zinc sulfate/Imidazole can be used for staining SDS-gels, but that does not really help my a lot. Does anybody have an idea how I might be able to keep everything in solution, seeing that the MD guys managed somehow...... I'm a bit desperate as I don't have many hits for this protein.
Cheers,
Chris B
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
----------
From: Mark J van Raaij
Hi Christopher,
you'll have to try to find out how they mix the components at MD and if they pH the solution afterwards or before (or not at all...) and to which pH. You can ask them; or try different mixing/pHing protocols on a small scale and see if one works in keeping all in solution.
Mark
Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
----------
From: Enrico Stura
When you give the composition of the screen condition, you must also give the conditions of
the protein buffer, since you get crystals when the two are mixed.
If you analyse many small salt crystals by SDS-PAGE you may still get staining since protein
will precipitate on the salt crystals.
Enrico.
Enrico A. Stura D.Phil. (Oxon) ,
----------
From: Mark J van Raaij
PS I would also try substituting imidazole for HEPES and zinc sulphate for zinc chloride and sodium sulphate.
----------
From: Roger Rowlett
Imidazole is basic. If you mix it directly with zinc salts you will get zinc hydroxide. Most screens are prepared by mixing stock solutions. In your case I would make up the screen from 1 M imidazole, pH 7.2, 1 M ZnSO4, and 50% PEG-4000. Add the PEG last. This should work. You may or may not need this specific salt or buffer to get crystals, and could swap them out or change concentration as required. 50 mM buffer may not be sufficient to control condition pH depending on the protein storage buffer composition.
Roger Rowlett
----------
From: Enrico Stura
The PEG could also be the problem, so you can mix your stock solutions by the method described
below and end up with the same result as soon as you add the PEG.
See:
Frances Jurnak: Effect of chemical impurities in polyethylene glycol on macromolecular crystallization
Journal of Crystal Growth
Volume 76, Issue 3, 2 August 1986, Pages 577-582
Enrico.
----------
From: Christopher Browning
Thanks for the replies. I think I got it figured out. The pH seems to be
the definitive factor. Indeed, the more alkaline the pH, the higher the
amount of precipitation. I just played a bit around with the pH of
Imidazole and it's cleared up. It also seemed to help to add things in
this order..... Imidazole--->water--->ZnSO4--->PEG.
Cheers,
Chris
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