Number of years ago Jaru Jancarik (the author of Screen I & II sold by HR)
while in Berkeley Structural Genomics Center (or may be even earlier) made an
observation regarding protein precipitation in condition A6 in that very
screen. Based on this observation HR sells now PCT (protein concentration test
or something similar). In brief, if your protein is concentrated enough and is
a regular protein, not a stellar, you will see medium to heavy precipitation.
In the case of proteins that are really of great quality you'll end up having
crystals in A6. After many years of experience I realized that A9 and A10 from
the same screen could be added to the list of conditions that are somewhat
indicative for choosing right concentration same way like Jaru did for A6.
My two Armenian drams worth,
Vaheh
________________________________
From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Roger
Rowlett
Sent: Thursday, July 19, 2012 4:42 PM
To: [email protected]
Subject: Re: [ccp4bb] Protein concentration vs Molecular wt...
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]<mailto:[email protected]>
On 7/19/2012 4:31 PM, [email protected]<mailto:[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]><mailto:[email protected]>
To: CCP4BB <[email protected]><mailto:[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.
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