Hi,
We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer.
Protein-to-well-solution ratio was 3:1.
When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio.
That is another important variable to test, I guess.
Regards,
-Andre.
________________________________________
From: CCP4 bulletin board [[log in to unmask]] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [[log in to unmask]]
Sent: Monday, February 27, 2012 1:25 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] Desalting columns
Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ?
For crystallization, the concentration needs to be
a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary)
b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen.
There is ample evidence for proteins crystallizing below 3 mg/ml.
The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins.
Sometimes the shape of a distribution matters ;-)
BR
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Sangeetha Vedula
Sent: Monday, February 27, 2012 8:02 AM
To: [log in to unmask]
Subject: [ccp4bb] Desalting columns
Dear bb users,
I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL.
I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums.
Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column.
Thanks a lot!
Best regards,
Sangeetha.
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