Dr E. Buxbaum
EB15 at le.ac.uk
Fri Nov 7 17:18:32 EST 1997
Peter Rozynek wrote:
> We have isolated a plant protein in efficient purity and amount which
> has been identified as important allergen and is also thought to play
> a important role in natural rubber synthesis. We´d like to analyze the
> 3-D structure of this protein. Any suggestions/hints/propasals for
> protein crystallization and or cooperation is greatly appreciated.
The first thing to do is to find conditions under which the protein
crystallizes. These then are optimized later.
For initial screening, place 10 ul protein solution (say 5-10 mg/ml) and
10 ul precipitating agent together in the middle of a silanized cover
slip. Fill a well of a 24 well plate with 1 ml of the precipitating
reagent and put some vaseline on the rim. Place the cover slip (drop
hanging from the underside) onto the well, so that you get a sealed
chamber, with the drop of protein solution hanging from the ceiling.
Keep for 2 weeks vibration free at constant temperature (usually 4
degrees C). During this time water will evaporate from the drop into the
liquid reservoir, until the concentrations in both are identical
(isothermic distillation). This gives you a slow increase of
precipitating agent, and hopefully crystalls. After these 2 weeks, check
for crystalls in the hanging drop under the microscope.
As precipitating agents you may try different concentrations in
different buffers (borate, tris, hepes, imidazole, acetate, citrate) of
ethanol, propanol, acetone, polyethylene glycol (of different MW),
ethylene glycol, ammonium sulfate, NaCl, MgSO4,....
Try them in combination too. There are sets of precipitation solution
available ready made, which simplifies the setup slightly. As you can
see, you are realy playing the numbers game here.
If you get crystalls under any of those conditions, try to optimise
them. Once you are that far, you may want to look for cooperation with
somebody with experience in X-ray crystallography. They will be able to
advice you on how to grow larger crystalls suitable for their maschines.
Oh, one more thing: Make sure you know how crystalls of your
precipitating agents look like. Protein and salt crystalls can be
distinguished by two methods:
a) A salt crystall, when crushed with a probe, will emit a grinding
sound. If you hear nothing, then you have just destroyed a perfectly
good protein crystall.
b) Protein crystalls readily take up dye, if that is added to the mother
liquor, because there is a lot of water space in them. In salt crystalls
the ions are tightly packed, the crystall won't take up the dye.
More information about the Proteins