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Protein Crystallography Forum Discuss research on protein crystallography and crystalization. Protein Crystallography Forum.


seaurchin and now?

seaurchin and now? - Protein Crystallography Forum

seaurchin and now? - Discuss research on protein crystallography and crystalization. Protein Crystallography Forum.


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Old 08-22-2012, 10:05 AM
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Default seaurchin and now?



Hey guys,
I'm new here in this forum and this is my first thread. I am trying to crystallize a protein which I isolated and purified myself from a fungus. This was a very tough and challenging process but now I got it proper pure and homogenous. I screened two crystallization kits and got one hit where I received sea-urchin crystals (see foto). The thing is that the amount of protein I got left is very limited, about 700 g. The condition was 30% (v/v) MPD, 0.1M Sodium cacodylat pH 6.5 and 0.2M Magnesium acetate (hanging drop, 0.5l protein solution (in 10mM HEPES pH 7.5) plus 0.5l mother liquid plus 1l water). It lasted kind of long that these urchins grew (1.5-2 weeks) at room temperature. Protein: 64 kDa, depending on buffer and chloride conz. sometimes monomeric or dimeric. Metallo-enzyme.
My question is now:
Can I be sure that these urchins are protein and not salt crystals?
Which parameter is the most promising to alter and in which direction (pH, additive, buffer, MPD, protein conz, Temp)?
Can I change buffer system from cacodylat (poisoning and expensive) to citrate, MES or BisTris?
What is a general origin for sea urchin an how can they be altered to bigger mono crystals?

Thanks in advance for your thoughts!
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