This is almost exactly our basic approach, too. Before we got a
dropsetter, we did 24 wells (1/2 screen) to get a feel for the correct
protein concentration. Some additional rules of thumb we use:
1. We usually start at 10 mg/mL protein and go up or down from there
depending on the results of the initial screen
2. If we observe a high frequency of precipitation in a 10 mg/mL
protein screen, we will usually set a 1/2 concentration screen by
diluting the screen solutions 1:1 with water. This frequently
uncovers additional hits in wells that were heavily precipitated in
the original screen. Empirically, proteins we study seem to
crystallize better in the higher protein/lower precipitant zone of
the phase diagram than the lower protein/higher precipitant zone. YMMV.
Cheers,
_______________________________________
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: [email protected]
On 7/19/2012 4:31 PM, [email protected] wrote:
I don't think there is such a rule, but in the old days, when we only
had Hampton Screen I and II, the rule was:
- Set up screen 1, look at the drops and you should expect some kind
of precipitation in 50% of the drops. If much less than that, increase
your protein concentration. If much more than that, decrease protein
concentration.
- Set up screen 2, look and expect 30% precipitation.
I used to cut corners and do the statistics at 1/2 of a screen (one
24-well plate). You can probably use this method to get within a
factor of 2 of the optimal concentration.
There are probably good statistics in the papers for the screens that
you may use. One of the advantages of structural genomics efforts is
that these things are known (and hopefully published).
Even older trick is to take a drop of protein and look under a
microscope, record how much AmSO4 it takes to cause precipitation. Do
the same with PEG. Keep adding a little at a time and look
immediately. This will give you an idea if you are near a reasonable
concentration. I think that this latter method does not tell you much
more than "physics-information" - which is how many zeroes there are:
whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable.
Mark
-----Original Message-----
From: james09 pruza <[email protected]>
To: CCP4BB <[email protected]>
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular
weight for crystallization trials of a soluble protein? Looking for
high molecular wt. protein ~50kDa.
James.
