Spherulites and PEG3350

From: Jan van Agthoven
Date: 24 August 2011 19:05


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, 

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From: Savvas Savvides
Date: 24 August 2011 19:40

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 <  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

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From: Gregory Verdon
Date: 24 August 2011 19:42



You should check these "spherulites" on SDS-PAGE gel to make sure that these contain your protein. Then you can start thinking about optimization.

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From: Regina Kettering
Date: 24 August 2011 19:46

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

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From: Jan van Agthoven
Sent: Wednesday, August 24, 2011 2:05 PM
Subject: [ccp4bb] spherulites and PEG3350

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From: Jan van Agthoven
Date: 24 August 2011 20:08



Thanks for the protocole and advice! I'll put my spherulites on gel. It will make things clear.

Jan

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From: Prince, D Bryan
Date: 24 August 2011 20:09

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 On Behalf Of Regina Kettering
Sent: Wednesday, August 24, 2011 2:47 PM

Subject: Re: [ccp4bb] spherulites and PEG3350

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From: Tom Peat
Date: 24 August 2011 22:48

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
Sent: Thursday, August 25, 2011 04:46 AM