I tried microbatch and the crystals are not stable. They dissolve overnight. I also have reproducibility issue. Can this be due to poor stability of adenosine?
Best
Maria
On Monday, May 5, 2014, Bob Cudney <[log in to unmask]> wrote:
> Try the microbatch first to see if the problem is related to ionic strength.
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> When possible and practical it is good to change only one variable at a time to identify cause and effect.
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> Kind Regards, Bob Cudney
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> Hampton Research
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> 34 Journey
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> Aliso Viejo, CA 92656-3317 USA
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> Telephone 1 949 425 1321 Extension 200
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> Fax 1 949 425 1611
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> E-mail [log in to unmask]
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> Web www.hamptonresearch.com
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> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of dusky dew
> Sent: Saturday, May 03, 2014 1:29 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] Crystals Disappearing Overnight
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> Thank you all for getting back!
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> I will set up the drops using microbatch method.
> Regarding the temp, I set them up in lab and put them in incubator. The lab temp may be slightly higher. So are they not stable at lower temp? Or its the shock?
> So how can I take care of the temp issue?
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> Thanks again!
> Maria
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> On Saturday, May 3, 2014, Bob Cudney <[log in to unmask]> wrote:
>> Hello Maria,
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>> Check to see if there might have been a temperature change between the time the crystals were present and when the crystals disappeared. If your sample has temperature dependent solubility, in this relatively low ionic strength condition, a temperature change could mean the difference between the presence and absence of crystals. That being said, if the experiment is returned to the temperature that produced the crystals, the crystals should/might reappear.
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>> If your drop is made by mixing 1 part of protein with 1 part of reagent the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v PEG 8,000, 50 mM Sodium cacodylate. Is this a vapor diffusion experiment? If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium cacodylate. The ionic strength of your drop would initially be higher than the ionic strength in your reservoir. This means water vapor leaves the reservoir and vapor diffuses into the drop, lowering the protein and reagent concentration in your drop. This decrease in relative supersaturation could dissolve a crystal. Your set up would be a reserve vapor diffusion. You say the crystals appeared right after setting the experiment so your crystallization is essentially a batch experiment. Therefore you might want to change your set up from a vapor diffusion to a microbatch experiment under oil. If you need more information about how to perform a microbatch experiment, let me know and I’ll explain.
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>> Hope this helps.
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>> Kind Regards, Bob Cudney
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>>
>>
>> Hampton Research
>>
>> 34 Journey
>>
>> Aliso Viejo, CA 92656-3317 USA
>>
>>
>>
>> Telephone 1 949 425 1321 Extension 200
>>
>> Fax 1 949 425 1611
>>
>> E-mail [log in to unmask]
>>
>> Web www.hamptonresearch.com
>>
>>
>>
>> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of dusky dew
>> Sent: Friday, May 02, 2014 4:39 AM
>> To: [log in to unmask]
>> Subject: [ccp4bb] Crystals Disappearing Overnight
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>> Dear All,
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>> I am trying to crystallize a protein with Adenosine. My protein is in 20 mM Tris, 300 mM NaCl and the crystals appear in a condition with 5 percent PEG8K, 0.1 M Sodium Cacodylate. The protein is incubated with adenosine for 1/2 hr before setting the drop. The crystals appear right after the drop is set but unfortunately they dissolve overnight. The plate is kept at 16 degree.
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>> Could anyone elaborate on this. Is it possibly occurring because Adenosine has stability issues.
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>> Thanks for your suggestions.
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>> ~ Maria
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