Re: [ccp4bb] The color of GFP crystals?
In my hands GFP fusions can produce colorless crystals (but still have GFP!) depending on the condition. Sometimes the crystals gradually become colorless with time - bleaching or perhaps some chemical effect - no idea. I've not tested the colorless crystals with UV to see if they still glow. Artem On Wed, Sep 29, 2010 at 3:41 AM, Tri Ngo wrote: > Dear CCP4 community, > > I wonder if anybody has experience about the color of GFP crystals. Is > there any chance that the GFP crystal will become colorless? We are working > on one membrane protein fused with GFP. Since we got some crystals which > didn't show any color (Condition 01), I am not sure if I should go for > optimization. > > Please check the link below for the image details: > http://www.docstoc.com/docs/55950590/GFP-crystals > > Because of the limited amount of sample, we used Topaz system (screening > chip) to do the screening and there is no way to confirm these crystals are > actually protein crystals. I noticed that the crystals are bigger day by > day. Is it a good sign to confirm the protein crystals? > > Thank you very much for your helpful information! > > Best wishes, > > * > TriNgo > > * > > Structural Biology Lab > > School of Medicine - Sungkyunkwan University > > Phone: 031-299-6150 > > >
Re: [ccp4bb] The color of GFP crystals?
Dear experts, Thank you very much for your input. Here are my answers for your questions: >To me, the first 3 pictures suggest that there is a faint greenish tinge in the little shapes (hopefully >crystalline) there. I guess the idea of the Topaz chip is that you can take it as is to the x-ray beam or am I >wrong? - I didn't see the greenish tinge in the original images. I think the color of the images was modified when I convert to pdf format. - The screening Topaz chip cannot apply for diffraction. They have a different chip for in-situ diffraction but we didn't use that chip in this case. >What GFP did you use, your pH is 4.6 or something around there according to your picture. If the >pH is true GFP will be colorless. You certainly have some more GFP lying around (without your >protein fused to it), just titrate it and see what pH dependency you have. - The protein was fused with GFPuv. It also shows the beautiful green color at the concentration 30mg/ml in the low pH buffer (20mM sodium acetate 4.6, 100mM NaCl, 0.2% Cymal5). Do you think that the crystal getting bigger is a good sign of protein crystal? My best regards, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
Re: [ccp4bb] The color of GFP crystals?
What GFP did you use, your pH is 4.6 or something around there according to your picture. If the pH is true GFP will be colorless. You certainly have some more GFP lying around (without your protein fused to it), just titrate it and see what pH dependency you have. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 29, 2010, at 4:41 AM, Tri Ngo wrote: > Dear CCP4 community, > > I wonder if anybody has experience about the color of GFP crystals. Is there > any chance that the GFP crystal will become colorless? We are working on one > membrane protein fused with GFP. Since we got some crystals which didn't show > any color (Condition 01), I am not sure if I should go for optimization. > > Please check the link below for the image details: > http://www.docstoc.com/docs/55950590/GFP-crystals > > Because of the limited amount of sample, we used Topaz system (screening > chip) to do the screening and there is no way to confirm these crystals are > actually protein crystals. I noticed that the crystals are bigger day by day. > Is it a good sign to confirm the protein crystals? > > Thank you very much for your helpful information! > > Best wishes, > > TriNgo > > Structural Biology Lab > School of Medicine - Sungkyunkwan University > Phone: 031-299-6150 >
Re: [ccp4bb] The color of GFP crystals?
Hi Tri, To me, the first 3 pictures suggest that there is a faint greenish tinge in the little shapes (hopefully crystalline) there. I guess the idea of the Topaz chip is that you can take it as is to the x-ray beam or am I wrong? Cheers, Boaz - Original Message - From: Tri Ngo Date: Wednesday, September 29, 2010 10:50 Subject: [ccp4bb] The color of GFP crystals? To: CCP4BB@JISCMAIL.AC.UK > Dear CCP4 community, > > I wonder if anybody has experience about the color of GFP > crystals. Is there > any chance that the GFP crystal will become colorless? We are > working on one > membrane protein fused with GFP. Since we got some crystals > which didn't > show any color (Condition 01), I am not sure if I should go for > optimization. > > Please check the link below for the image details: > http://www.docstoc.com/docs/55950590/GFP-crystals > > Because of the limited amount of sample, we used Topaz system > (screeningchip) to do the screening and there is no way to > confirm these crystals are > actually protein crystals. I noticed that the crystals are > bigger day by > day. Is it a good sign to confirm the protein crystals? > > Thank you very much for your helpful information! > > Best wishes, > > * > TriNgo > > * > > Structural Biology Lab > > School of Medicine - Sungkyunkwan University > > Phone: 031-299-6150 > Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan
[ccp4bb] The color of GFP crystals?
Dear CCP4 community, I wonder if anybody has experience about the color of GFP crystals. Is there any chance that the GFP crystal will become colorless? We are working on one membrane protein fused with GFP. Since we got some crystals which didn't show any color (Condition 01), I am not sure if I should go for optimization. Please check the link below for the image details: http://www.docstoc.com/docs/55950590/GFP-crystals Because of the limited amount of sample, we used Topaz system (screening chip) to do the screening and there is no way to confirm these crystals are actually protein crystals. I noticed that the crystals are bigger day by day. Is it a good sign to confirm the protein crystals? Thank you very much for your helpful information! Best wishes, * TriNgo * Structural Biology Lab School of Medicine - Sungkyunkwan University Phone: 031-299-6150
