Re: [ccp4bb] cryoprotectant
Hi Reza, Check the following reference: Cryoprotection properties of salts of organic acids: a case study for a tetragonal crystal of HEW lysozyme. Bujacz G, Wrzesniewska B, Bujacz A. Acta Crystallogr D Biol Crystallogr. 2010 Jul;66(Pt 7):789-96. Cheers, Karolina W dniu 2014-12-01 10:59, Reza Khayat napisał(a): > Hi, > > Has anyone used citrate as the sole cryoprotectant? If so, > what concentration was needed? > > Best wishes, > Reza > > Reza Khayat, PhD > Assistant Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > www.khayatlab.org [1] Links: -- [1] http://www.khayatlab.org
Re: [ccp4bb] cryoprotectant
Hi Reza, 1.0 and 1.25 M citrate are potential cryo solutions. They still had faint ice rings present but were not dominant. It is also difficult to make a solution more concentrated then 1.75 M citrate. Hope this helps. -Adam > On Dec 1, 2014, at 10:59 AM, Reza Khayat wrote: > > Hi, > > Has anyone used citrate as the sole cryoprotectant? If so, > what concentration was needed? > > Best wishes, > Reza > > Reza Khayat, PhD > Assistant Professor > The City College of New York > Department of Chemistry, MR-1135 > 160 Convent Avenue > New York, NY 10031 > Tel. (212) 650-6070 > www.khayatlab.org
[ccp4bb] cryoprotectant
Hi, Has anyone used citrate as the sole cryoprotectant? If so, what concentration was needed? Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
80% saturated lithium sulfate should have about the correct ionic strength to match your crystallization conditions. The crystals need to be transfered with as little mother liquor as possible to avoid lithium phosphate crystallization. Robert Kirchdoerfer suggestion is also excellent, but careful about matching the pH. Enrico. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
I don't know how many of these crystals you have, but if you can spare one try freezing it straight out of the drop without cryoprotection. Certain salts can act as cryoprotectants at high enough concentrations. I don't know about phosphate salts, but I've had crystals that grew in 2.5M ammonium sulfate which I froze without cryoprotection and rarely saw an ice ring in my diffraction. Good luck, Mike - Original Message - From: "Jerry McCully" To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, December 15, 2010 1:13:09 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate Dear All; Recently we got some crystals from the condition #51 in the SaltRx crystallization kit from Hampton research. It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test diffraction ASAP. What cryoprotectant did you use for this condition? Thanks a lot and have a nice holiday season! Jinghua -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
Quickly passing the crystal through Paratone N has worked well for me when I crystallize in ammonium sulfate or sodium citrate conditions. Another trick is to dissolve sucrose (table sugar) in 10uL of the reservoir solution until it is saturated. Then separate the sucrose-reservoir mix into two 5ul drops. Add 5ul of the reservoir solution to one of the drops to make a 50% solution. Pass the crystal through the 50% saturated sucrose solution, then the 100% saturated sucrose solution and freeze. Good luck with the crystal! Bryan -- 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. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robert Kirchdoerfer Sent: Wednesday, December 15, 2010 4:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate I've had good luck cryoprotecting high salt crystal conditions with sodium malonate (2.0-2.5M). Start with a 5M sodium malonate solution and dilute to 40-50% with mother liquor. good luck, Rob From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully [for-crystallizai...@hotmail.com] Sent: Wednesday, December 15, 2010 1:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate Dear All; Recently we got some crystals from the condition #51 in the SaltRx crystallization kit from Hampton research. It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test diffraction ASAP. What cryoprotectant did you use for this condition? Thanks a lot and have a nice holiday season! Jinghua
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
I've had good luck cryoprotecting high salt crystal conditions with sodium malonate (2.0-2.5M). Start with a 5M sodium malonate solution and dilute to 40-50% with mother liquor. good luck, Rob From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully [for-crystallizai...@hotmail.com] Sent: Wednesday, December 15, 2010 1:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate Dear All; Recently we got some crystals from the condition #51 in the SaltRx crystallization kit from Hampton research. It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test diffraction ASAP. What cryoprotectant did you use for this condition? Thanks a lot and have a nice holiday season! Jinghua
Re: [ccp4bb] cryoprotectant for protein crystal grown from Di-sodium hydrogen phosphate
Mother liquor plus 30% glycerol or 30% glucose will cryoprotect pretty much anything, if it does not cause crystal cracking. We have had very good general luck with 25-30% glucose, and it's easy to prepare from your well solution or crystallization master mix. Add 150 mg of glucose to a microcentrifuge tube, make up to the 0.5 mL mark with your crystallization well solution, sonicate or otherwise mix thoroughly until dissolved. You can either transfer and swish crystals in the cryo solution, or gradually dilute your drop to 10X volume of the cryo-solution. Cheers. On 12/15/2010 4:13 PM, Jerry McCully wrote: Dear All; Recently we got some crystals from the condition #51 in the SaltRx crystallization kit from Hampton research. It contains 1.5M Na2HPO4 and 0.1M Tris(pH8.5). We am going to do a test diffraction ASAP. What cryoprotectant did you use for this condition? Thanks a lot and have a nice holiday season! Jinghua -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu
[ccp4bb] Cryoprotectant for jaffamine - summary
Hi all, Thanks for the reply. I have ultimately used 15% glycerol as cryo. It worked well. I referred to this paper McFerrin and Snell, Cryoprotectant quantification J. Appl. Cryst. (2002). 35, 538±545. Now setting up grid with different concentrations of jeffamine. Arpita Below is a summary of messages I received; Hi.. Sorry for the non-ccp4 query.I have a protein crystal growing at 0.1 M HEPES-Na (pH7.5), 20% Jeffamine M-600 (sitting drop). Can anyone please suggest for the cryprotectant to be used? Arpita. Hi Arpita, do you work in the group of Shekhar Mande? if yes then can you please convey my regards to him.well anyways coming to your question, you can use Jeffamine itself as a good cryo protectant.. i had recently used 45 % jeffamine as an effective cryo. but you should be careful that direct transfer to 45 % jeffamine "might" destroy the diffraction of the crystal what i will be tempted to do is slowly increasing the concentration of jeffamine.. you can try doing both by direct transfer or by equlibration..do let me know if you have any doubts... regards Avinash hi.. thank u 4 the reply...ya..i m working with him ...i will surely convey to him..what is equilibration?. actually i do not have too many crystals in that condition. can I set up a grid upto 45% jeffamine? Arpita yes you can and i will recommend you to use online grid make "SAmBA".. if you cannot find the link then you can get it from my web-page (interactive tools... my web page link is http://www.freewebs.com/avikale/ please feel free to ask any question if you might have regards Avinash Arpita, Try this. Cryoprotectant database for protein crystals. http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html best Yogi Jeffamine itself is a cryo. Simply raise its concentration, Dima jeffamine itself might be a cryo. jan Since Jeffamine M-600 is a small polymer, I believe you can freeze your crystals directly. If required you may have to increase the Jeffamine conc'n to 25-30% (Let me know if it worked). Anthony I think you can use the crystals as they are or increase jeffamine to 30%. Never had jeffamine but I think it is like PEG400. Jacqueline
[ccp4bb] Cryoprotectant for jaffamine
Hi.. Sorry for the non-ccp4 query.I have a protein crystal growing at 0.1 M HEPES-Na (pH7.5), 20% Jeffamine M-600 (sitting drop). Can anyone please suggest for the cryprotectant to be used? Arpita.
Re: [ccp4bb] cryoprotectant for 1,6 hexanediol
On Thu, 2 Apr 2009, HanJie_Heng Chiat Tai wrote: I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH 6.5). What's the cryoprotectant can be used to flash cool this crystal? I recently grew crystals in 0.3M 1,6.hexanediol, cryoprotectant was PEG 400 (final conc. 32 %). Proteins 2009; 75:748.759. Kaj
Re: [ccp4bb] cryoprotectant for 1,6 hexanediol
If you are uncertain, just freeze your buffer :-) Very old trick from the stone age of crystallography. Long time ago ~ 1999 I cryoed one crystal with 1.5 M hexanediol but there was also 10% glycerol around. Jürgen On 2 Apr 2009, at 14:56, HanJie_Heng Chiat Tai wrote: Hi, Jim, What's the concentration? I know that [hexanediol] between 2.5 - 3.4 M no additional cryoprotectant is required. But in my case my hexanediol conc is only 2.1 M Rgds, HengChiat Tai (HanJie) > Date: Thu, 2 Apr 2009 13:11:27 -0500 > From: jim.pflugr...@rigaku.com > To: chemtai2...@hotmail.com > CC: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] cryoprotectant for 1,6 hexanediol > > The cryoprotectant is 1,6 hexanediol. > > Jim > > On Thu, 2 Apr 2009, HanJie_Heng Chiat Tai wrote: > > > > > > > Hi, > > > > > > > > I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH 6.5). > > > > > > > > What's the cryoprotectant can be used to flash cool this crystal? > > > > > > > > Any online protein crystal cryoprotectant database or published literature available I can check with to determine to type and concentration of the cryoprotectant used for my crystal. > > > > > > HengChiat Tai > > > > > > > > _ > > Rediscover Hotmail®: Get e-mail storage that grows with you. > > http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Storage1_042009 Rediscover Hotmail®: Get quick friend updates right in your inbox. Check it out. - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Biochemistry and Molecular Biology, W8708 615 North Wolfe Street Baltimore, MD 21205 Phone: +1-410-614-4742 Fax: +1-410-955-3655
Re: [ccp4bb] cryoprotectant for 1,6 hexanediol
Hi, Jim, What's the concentration? I know that [hexanediol] between 2.5 - 3.4 M no additional cryoprotectant is required. But in my case my hexanediol conc is only 2.1 M Rgds, HengChiat Tai (HanJie) > Date: Thu, 2 Apr 2009 13:11:27 -0500 > From: jim.pflugr...@rigaku.com > To: chemtai2...@hotmail.com > CC: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] cryoprotectant for 1,6 hexanediol > > The cryoprotectant is 1,6 hexanediol. > > Jim > > On Thu, 2 Apr 2009, HanJie_Heng Chiat Tai wrote: > > > > > > > Hi, > > > > > > > > I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH > > 6.5). > > > > > > > > What's the cryoprotectant can be used to flash cool this crystal? > > > > > > > > Any online protein crystal cryoprotectant database or published literature > > available I can check with to determine to type and concentration of the > > cryoprotectant used for my crystal. > > > > > > HengChiat Tai > > > > > > > > _ > > Rediscover Hotmail®: Get e-mail storage that grows with you. > > http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Storage1_042009 _ Rediscover Hotmail®: Get quick friend updates right in your inbox. http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Updates1_042009
Re: [ccp4bb] cryoprotectant for 1,6 hexanediol
The cryoprotectant is 1,6 hexanediol. Jim On Thu, 2 Apr 2009, HanJie_Heng Chiat Tai wrote: Hi, I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH 6.5). What's the cryoprotectant can be used to flash cool this crystal? Any online protein crystal cryoprotectant database or published literature available I can check with to determine to type and concentration of the cryoprotectant used for my crystal. HengChiat Tai _ Rediscover Hotmail?: Get e-mail storage that grows with you. http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Storage1_042009
[ccp4bb] cryoprotectant for 1,6 hexanediol
Hi, I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH 6.5). What's the cryoprotectant can be used to flash cool this crystal? Any online protein crystal cryoprotectant database or published literature available I can check with to determine to type and concentration of the cryoprotectant used for my crystal. HengChiat Tai _ Rediscover Hotmail®: Get e-mail storage that grows with you. http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Storage1_042009
Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal
I think we still have better luck with longer, slower, more gentle soaks - but its crystal-dependent. Try raising the [PEG] at the same time as you raise the [glycerol]. Phoebe Original message >Date: Fri, 31 Oct 2008 18:57:53 -0400 >From: Artem Evdokimov <[EMAIL PROTECTED]> >Subject: Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal >To: CCP4BB@JISCMAIL.AC.UK > >Why soak for a whole minute? A single pass through cryo is usually enough, >and that takes a couple of seconds with the right set-up... > >You could try oil - if you're lucky it solves your issues. Note that not all >oils are the same, and many people succeed with blended compositions rather >than pure stuff. > >Finally, you could always try my humble recipe: > >http://www.xtals.org/crystal_cryo.pdf > >Good luck, > >Artem > >-Original Message- >From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E >rajakumar >Sent: Friday, October 31, 2008 5:22 PM >To: CCP4BB@JISCMAIL.AC.UK >Subject: [ccp4bb] Cryoprotectant for protein-DNA complex crystal > >Dear All >I am working on protein-DNA complex crystals for data >collection. These crystals are grown in 15-20 % of >PEG3350 or PEG4000 with pH of 6 to 7. When I soak the >crystals more than a minute in the cryo solution >(15-20% of Glycerol or ethylenglycol + reservoir) the >resolution of diffraction is becoming weak (reducing >to 6.0 A from 4.5 A) and also the spots are getting >spread (increase in mosaicity). Appears that Glycerol >or Ethylene glycol not good cryoprotectants in this >case. Is there any study on effect of cryoprotectant >on protein-DNA complex crystal and protein-DNA complex >dissociation? I also want to know which type >(organics, oils, polyols, sugars, polymers.) of >cryoprotectant is most preferred in protein-DNA >complex crystal. >Thanking you in advance >Rajakumara > > >E. Rajakumara >Postdoctoral Fellow > Strcutural Biology Program > Memorial Sloan-Kettering Cancer Center > New York-10021 > NY > 001 212 639 7986 (Lab) > 001 917 674 6266 (Mobile) > > > > Get your new Email address! >Grab the Email name you've always wanted before someone else does! >http://mail.promotions.yahoo.com/newdomains/aa/ Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal
Hi Rajakumara, You might try growing the crystals at say 25-30% of your PEG, it might be enough to cryofreeze without additional cryoprotectant if the current mother liquor is not enough. You may also try a serial soak in multiple steps increasing the cryo conc in steps of 3% or so to make the transition gentle. This would be in addition to a 2-step soak as mentioned in Artem's document. If your diffraction is 4.5A-6.0A due to very small complex crystals and the resolution deterioration is due to x-ray exposure, then you might try seeding to get larger crystals which may be able to tolerate better the exposure to cryoprotectant or x-rays. Although none of these answer your original question, it may help. Best, Debanu. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Artem Evdokimov Sent: Friday, October 31, 2008 3:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal Why soak for a whole minute? A single pass through cryo is usually enough, and that takes a couple of seconds with the right set-up... You could try oil - if you're lucky it solves your issues. Note that not all oils are the same, and many people succeed with blended compositions rather than pure stuff. Finally, you could always try my humble recipe: http://www.xtals.org/crystal_cryo.pdf Good luck, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Friday, October 31, 2008 5:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotectant for protein-DNA complex crystal Dear All I am working on protein-DNA complex crystals for data collection. These crystals are grown in 15-20 % of PEG3350 or PEG4000 with pH of 6 to 7. When I soak the crystals more than a minute in the cryo solution (15-20% of Glycerol or ethylenglycol + reservoir) the resolution of diffraction is becoming weak (reducing to 6.0 A from 4.5 A) and also the spots are getting spread (increase in mosaicity). Appears that Glycerol or Ethylene glycol not good cryoprotectants in this case. Is there any study on effect of cryoprotectant on protein-DNA complex crystal and protein-DNA complex dissociation? I also want to know which type (organics, oils, polyols, sugars, polymers.) of cryoprotectant is most preferred in protein-DNA complex crystal. Thanking you in advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your new Email address! Grab the Email name you've always wanted before someone else does! http://mail.promotions.yahoo.com/newdomains/aa/
Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal
Try soaking for about 5 seconds. You should only need to coat the surface of the crystal to cryoprotect it. Bill On Oct 31, 2008, at 2:22 PM, E rajakumar wrote: When I soak the crystals more than a minute
Re: [ccp4bb] Cryoprotectant for protein-DNA complex crystal
Why soak for a whole minute? A single pass through cryo is usually enough, and that takes a couple of seconds with the right set-up... You could try oil - if you're lucky it solves your issues. Note that not all oils are the same, and many people succeed with blended compositions rather than pure stuff. Finally, you could always try my humble recipe: http://www.xtals.org/crystal_cryo.pdf Good luck, Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of E rajakumar Sent: Friday, October 31, 2008 5:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Cryoprotectant for protein-DNA complex crystal Dear All I am working on protein-DNA complex crystals for data collection. These crystals are grown in 15-20 % of PEG3350 or PEG4000 with pH of 6 to 7. When I soak the crystals more than a minute in the cryo solution (15-20% of Glycerol or ethylenglycol + reservoir) the resolution of diffraction is becoming weak (reducing to 6.0 A from 4.5 A) and also the spots are getting spread (increase in mosaicity). Appears that Glycerol or Ethylene glycol not good cryoprotectants in this case. Is there any study on effect of cryoprotectant on protein-DNA complex crystal and protein-DNA complex dissociation? I also want to know which type (organics, oils, polyols, sugars, polymers.) of cryoprotectant is most preferred in protein-DNA complex crystal. Thanking you in advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your new Email address! Grab the Email name you've always wanted before someone else does! http://mail.promotions.yahoo.com/newdomains/aa/
[ccp4bb] Cryoprotectant for protein-DNA complex crystal
Dear All I am working on protein-DNA complex crystals for data collection. These crystals are grown in 15-20 % of PEG3350 or PEG4000 with pH of 6 to 7. When I soak the crystals more than a minute in the cryo solution (15-20% of Glycerol or ethylenglycol + reservoir) the resolution of diffraction is becoming weak (reducing to 6.0 A from 4.5 A) and also the spots are getting spread (increase in mosaicity). Appears that Glycerol or Ethylene glycol not good cryoprotectants in this case. Is there any study on effect of cryoprotectant on protein-DNA complex crystal and protein-DNA complex dissociation? I also want to know which type (organics, oils, polyols, sugars, polymers ) of cryoprotectant is most preferred in protein-DNA complex crystal. Thanking you in advance Rajakumara E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) Get your new Email address! Grab the Email name you've always wanted before someone else does! http://mail.promotions.yahoo.com/newdomains/aa/
Re: [ccp4bb] cryoprotectant for crystals in isopropanol
Another anecdote for you: I had crystals that grew in 15% PEG 2000 and 15% isopropanol. Cryos with glycerol melted the crystals. The best cryo I found was with 15% PEG 2000, 15% isopropanol, and 20% PEG 400. It's hard to predict how your crystals will behave with different cryoprotectants-- hopefully you have enough crystals to try several different conditions. Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of shivesh kumar Sent: Wednesday, March 19, 2008 3:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryoprotectant for crystals in isopropanol Dear all, The question is regarding the cryoprotectant.We have crystallized a protein in 20% PEG 500 with 10% isopropanol.What should be the cryo we should try for data collection.Also,what percentage of PEG 500 is sufficient enough as a cryo for data collection. We are also trying to crystallize it with higher PEG's also like 1K, 2K, 4K.Lets C.Since the condition is having isopropanol also and it is reported that mounting with isoprop is very difficult and it evaporates as soon as we open the slides. Thanx in advance... Shivesh Kumar
Re: [ccp4bb] cryoprotectant for crystals in isopropanol
Not that all proteins are created alike, but we had a protein crystallize in a similar situation (PEG/20%IPA). I don't remember the exact details, but we did have good luck using just glycerol as a cryoprotectant. That is, I made up the well buffer and added to that well buffer 20-25% glycerol (in addition to IPA/PEG), and moved the crystal into that. We had no issues with cracking or anything, and data collection went well. The first thing I tried worked, so I don't have a lot of trial information for you. You could probably reduce the glycerol a bit with no worries, but I don't know. Good luck Dave shivesh kumar wrote: Dear all, The question is regarding the cryoprotectant.We have crystallized a protein in 20% PEG 500 with 10% isopropanol.What should be the cryo we should try for data collection.Also,what percentage of PEG 500 is sufficient enough as a cryo for data collection. We are also trying to crystallize it with higher PEG's also like 1K, 2K, 4K.Lets C.Since the condition is having isopropanol also and it is reported that mounting with isoprop is very difficult and it evaporates as soon as we open the slides. Thanx in advance... Shivesh Kumar
[ccp4bb] cryoprotectant for crystals in isopropanol
Dear all, The question is regarding the cryoprotectant.We have crystallized a protein in 20% PEG 500 with 10% isopropanol.What should be the cryo we should try for data collection.Also,what percentage of PEG 500 is sufficient enough as a cryo for data collection. We are also trying to crystallize it with higher PEG's also like 1K, 2K, 4K.Lets C.Since the condition is having isopropanol also and it is reported that mounting with isoprop is very difficult and it evaporates as soon as we open the slides. Thanx in advance... Shivesh Kumar
