Dear All,
Thank you very much for all your
suggestions, I guess I will busy for some time now. I have list out the
suggestions I got for whom might be interested.
Improving crystal shapes:
l Add 10-20% organic solvent (menthol, ethanol, propanol, butanol, acetone,
etc) to crystallization buffer
l Additive screen
l Change temperature for crystallization
l Change crystallization buffer pH, for example, Na-acetate (pH4.6),
Na-cacodylate (pH5.5), Bis-tris (pH6.6), Tris-HCL (pH7.5), Bis-Tris Propane
(pH8.5), etc.
To determine either the crystals are
protein or not:
l Loop out a cluster of crystals to confirm by SDS PAGE, but need
intensive cleaning
l Confirm by Itiz dye
Future improvements:
l Improve cryo for better diffraction, can also test RT diffraction.
Again, thank you all very much, all your
advices are very much appreciated.
Kind regards
William
Date: Fri, 11 Apr 2014 03:02:51 +0100
From: williamlee0...@hotmail.com
Subject: Re: [ccp4bb] suggestion on improving sea urchin like crystals
To: CCP4BB@JISCMAIL.AC.UK
Here is some information regarding to my
crystals.
Crystallization condition:
0.9M-1.7M Ammonium Tartrate Dibasic
50mM-150mM Bis-Tris Propane (pH7.0 &
6.5) / 50mM-150mM Tris (pH7.5 & 8.5)
Protein buffer contains 50mM ADA, pH6.5,
100mM NaCl, 10mM B-ME
Regards
William
Date: Fri, 11 Apr 2014 01:50:18 +0100
From: williamlee0...@hotmail.com
Subject: [ccp4bb] suggestion on improving sea urchin like crystals
To: CCP4BB@JISCMAIL.AC.UK
Dear All,
I am currently working on a ligand bound
protein complex. From my initial crystallization screens I have identified a
condition which gives me sea urchin like crystals. I managed to repeat these
crystals in my optimization conditions, in fact I can see crystals in all the
wells but there is no significant difference between them. All conditions give
me similar size and amount of crystals. To confirm these are protein crystals
or not, I tried expose the crystals to X-ray beam (in-house). Good news is that
I have no obvious salt diffraction at high resolution, but the bad news is my
low resolution diffractions are not really believable. In addition, these
crystals seem to be quite easy to separate into needles but they are too small
to tell if they do crack like most the protein crystals.
I have attached a picture of my crystals
and the diffraction pattern I got at low resolution.
I am hoping if anyone can suggest me on how
to improve or change the shape of these crystals if they are genuine protein
crystals.
Many thanks Kind regards
William
