Re: [ccp4bb] spherulites and PEG3350
Dear Jan I would recommend running the following protocol on your spherulites. Just pretend that they are crystals :) This was posted some time ago on the ccp4bb. best regards Savvas On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete kenneth.verstra...@ugent.be wrote: Hi Ivan, there are several tests (e.g. Izit dye, crush test) you can do discern protein from salt crystals but what was always very informative to me (and certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the crystals using the following protocol: - select a drop which contains some substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother liquor containing a 10% higher concentration of precipitant) - transfer all the crystalline material from the drop into the PCR-tube using a pipet (use stabilizing buffer from the PCR tube to collect all crystals) - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope. They should be at the bottom of the PCR-tube. - Remove as much as supernatant as you can (make sure not to remove your crystals), add stabilizing buffer to wash the crystals, and centrifuge again - repeat this washing protocol a few times - after the final washing step, add Laemli-buffer to the crystals and use this sample to load the SDS-PAGE gel - include a positive (eg. solubilize another drop directly in Laemli-buffer) and a negative (final washing buffer) control - use silver staining to visualize the protein This always works for me. If you don't see a band at this point I would be worried that it is salt. You could then choose to do a Western blot instead of silver staining to increase the sensitivity. Make sure to include control samples then. Kind regards, Kenneth Verstraete L-PROBE Ghent University Belgium On 24 Aug 2011, at 20:05, Jan van Agthoven wrote: Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks,
Re: [ccp4bb] spherulites and PEG3350
You should check these spherulites on SDS-PAGE gel to make sure that these contain your protein. Then you can start thinking about optimization. On Wed, Aug 24, 2011 at 2:05 PM, Jan van Agthoven janc...@gmail.com wrote: Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks,
Re: [ccp4bb] spherulites and PEG3350
Thanks for the protocole and advice! I'll put my spherulites on gel. It will make things clear. Jan 2011/8/24 Regina Kettering reginaketter...@yahoo.com Something to consider is the quality of the PEG 3350. We have found that different qualities of PEG 3350 can give different results, depending on the type and amount of contaminants. What used to be the Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax). We use high quality PEG 3350 for normal screening, but switch to the highest quality grade we can get for optimizing. Regina -- *From:* Jan van Agthoven janc...@gmail.com *To:* CCP4BB@JISCMAIL.AC.UK *Sent:* Wednesday, August 24, 2011 2:05 PM *Subject:* [ccp4bb] spherulites and PEG3350 Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks,
Re: [ccp4bb] spherulites and PEG3350
Something else to try would be the Protic Ionic Liquid kit from Hampton. I recently had crystals of a protein that would only grow as laminated stacks of plates. Optimizing the conditions and using an additive screen didn't improve crystal morphology. I tried the PIL kit from Hampton and was able to get single, thick plates in several conditions. At the ACA meeting in Hawaii a few years back, there was a poster about ageing your PEG solutions by microwaving them and letting them cool on the bench. This was the only way that the poster's author could get reproducible crystals of her target protein. Good Luck! Bryan From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Regina Kettering Sent: Wednesday, August 24, 2011 2:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] spherulites and PEG3350 Something to consider is the quality of the PEG 3350. We have found that different qualities of PEG 3350 can give different results, depending on the type and amount of contaminants. What used to be the Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax). We use high quality PEG 3350 for normal screening, but switch to the highest quality grade we can get for optimizing. Regina From: Jan van Agthoven janc...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, August 24, 2011 2:05 PM Subject: [ccp4bb] spherulites and PEG3350 Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks, -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] spherulites and PEG3350
You might to consider that PEG 3350 has phosphate contamination, so playing around with small amounts of phosphate (or removing it) might be worthwhile. Cheers, tom From: Regina Kettering [mailto:reginaketter...@yahoo.com] Sent: Thursday, August 25, 2011 04:46 AM To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] spherulites and PEG3350 Something to consider is the quality of the PEG 3350. We have found that different qualities of PEG 3350 can give different results, depending on the type and amount of contaminants. What used to be the Fluka PEG 3350 is now the pharm grade of PEG 3350 (aka Miralax). We use high quality PEG 3350 for normal screening, but switch to the highest quality grade we can get for optimizing. Regina From: Jan van Agthoven janc...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, August 24, 2011 2:05 PM Subject: [ccp4bb] spherulites and PEG3350 Dear all, I recently obtained some spherulites while trying to crystallize my protein. The spherulites are manually reproducible, but changing pH, protein concentration, and salt concentration does not result in crystal formation. Microseeding with crushed spherulites isn't a solution either as it only yields new spherulites. Next stepp is the use of an optimization kit but I have a limited amount of material, and I start doubting that these are protein spherulites, as the spherulites are not particularly soft. The condition contains 15% PEG 3350 and 200 mM NaCl. Does anyone know if PEG 3350 forms easily spherulites around that concentration? Thanks,
