See below. I believe Ray intended this to be sent to the entire bb and especially to Subbu!
And in reading Ray's message, I am reminded that I forgot to mention what he may be hinting at. You should also try to optimize by creating a fine grid screen around your identified condition varying in particular pH and precipitant concentration.
best,
Eric
On Thu, 21 Jul 2011, [log in to unmask] wrote:
>What is the pI? Perhaps the protein will be more soluble close to this pH? You could increase the salt and/or add some glycerol or a
>cofactor?
>
>Normally crystallization is most affected by the pH, 2 - 10% glycerol or temperature 4 - 25 degrees C.
>Cheers
>
>Ray Brown
>
>_____________________________________________________________________________________________________________________________________
>From: Eric Larson <[log in to unmask]>
>To: [log in to unmask]
>Sent: Thu, July 21, 2011 1:40:51 PM
>Subject: Re: [ccp4bb] Concentrating a protein solution - subbu
>
>Hi Subbu,
>
>You got crystals at 1mg/ml so you probably don't need to concentrate your protein any higher, especially since you suggest that
>concentrating beyond that is problematic. Instead, you may want to try to optimize the crystallization condition you have already
>identified. Some possible things to try: additive screen, include specific ligands, different temperature, different ratios of
>protein solution to crystallization solution, seeding, different crystallization methods (hanging vs. sitting drop, batch,
>diffusion,...), ...
>
>good luck,
>
>Eric
>
>================================
>Eric T. Larson, PhD
>Biomolecular Structure Center
>Department of Biochemistry
>Box 357742
>University of Washington
>Seattle, WA 98195
>
>email: [log in to unmask]
>================================
>
>On Thu, 21 Jul 2011, Narayanan Ramasubbu wrote:
>
>> Dear All:
>> We have been trying to crystallize a protein which is large - > 100 kDa. This is soluble but the best we can get is about 1 mg/mL.
>> It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether
>there are methods that others use to increase the concentration other that using amicon columns.
>> Any help will be appreciated.
>> Thanks
>> Subbu
>>
>
>
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