Re: [ccp4bb] where I have been going wrong in crystallization?
Hi Martyn, When it comes to micro-dialysis, you could check the following paper: http://www.jbc.org/content/260/25/13580.full.pdf+html (freely available, check in particular the appendix for the micro-dialysis setup that was used, that was for heat-labile enterotoxin) Fred. PS I know this is not exactly answering your question but still you might find this setup useful MARTYN SYMMONS wrote: Thanks to those who replied. The humour comes of course from a slight feeling that there is some truth in the 'respectful' version. Proteins are wonderful subtle machines so perhaps we should be careful and thoughtful when we expose them to the horrors of most crystallization conditions! Which takes me to the original point of my search - which was for a dialysis method for lysozyme. I was trying to set up a micro dialysis format for my protein but wanted to test it first. I am using spectrapore 8K dialysis membrane and wondered if that would retain lysozyme enough to get crystals (I'd like to stick with this cut-off as the physical and sealing properties of other membranes might be different). Or if someone has a higher MW cheap alternative protein/condition? Best wishes - agus 'Bliadhna Mhath Ur Dhuibh Uile' as we say in gaelic. Martyn - Martyn Symmons Cambridge
Re: [ccp4bb] where I have been going wrong in crystallization?
Thanks to those who replied. The humour comes of course from a slight feeling that there is some truth in the 'respectful' version. Proteins are wonderful subtle machines so perhaps we should be careful and thoughtful when we expose them to the horrors of most crystallization conditions! Which takes me to the original point of my search - which was for a dialysis method for lysozyme. I was trying to set up a micro dialysis format for my protein but wanted to test it first. I am using spectrapore 8K dialysis membrane and wondered if that would retain lysozyme enough to get crystals (I'd like to stick with this cut-off as the physical and sealing properties of other membranes might be different). Or if someone has a higher MW cheap alternative protein/condition? Best wishes - agus 'Bliadhna Mhath Ur Dhuibh Uile' as we say in gaelic. Martyn - Martyn Symmons Cambridge - Original Message From: David Briggs To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 21 December, 2009 17:26:15 Subject: Re: [ccp4bb] where I have been going wrong in crystallization? Hi Martyn, this recipe tends to work for me... Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.8 Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.8 Additional Reagents: Index Reagent 8, 22, 28, 31, 34, 40, 58, 59, 69, 86, 88 Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or vapor diffusion works fine. ==> Direct copy/paste from http://hamptonresearch.com/experiments.aspx <== HTH and Happy Christmas. Cheers, Dave David C. Briggs PhD University of Manchester E-mail: david.c.bri...@manchester.ac.uk Twitter: @xtaldave Skype: DocDCB 2009/12/21 MARTYN SYMMONS : > Dear All > checking out the Lysozyme crystallization methods on the web I liked the > Rigaku Instructions that I found: > (http://www.rigaku.com/protein/crystallization.html) > > "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, > respectfully, for a total drop size of 6ul..." > > So perhaps sometimes I am just not respectful enough to deserve crystals ? > >good wishes to all > regards, > Martyn > --- > Martyn Symmons > MRC-MBU Cambridge UK > 'Chan fhiosrach mur feòraich.' > Gaelic proverb - > Nothing asked, nothing learned. >
Re: [ccp4bb] where I have been going wrong in crystallization?
Maybe is a mistake by a non-English speaker. Instead of "respectfully" should say "respectively"
Re: [ccp4bb] where I have been going wrong in crystallization?
It makes slight sense if the word 'respect' can be correlated with whether the drop is mixed (protein+ppt). i have heard some protein may indeed crystallize if it were not mixed or vice-versa (do not expect it with lysozyme though). it would make sense if diffusion played a role but with few ul drop (the tip of pipette covering the surface of the drop in 96-well), i imagine what would be the real reason/advantage for this argument of mixing vs not mixing drops. -- Karthik On Mon, Dec 21, 2009 at 4:19 PM, Felix Frolow wrote: > If "respect" is concerned, try to stop a blow of a sharp samurai sword with > a sharp word. > I myself have no problems whatsoever with the Rigaku recipes. And yes, I > have stopped, > at leas once, a blow of samurai sword with my bare hands. Since than I > paint pictures with my left foot. > BTW > In ALL my crystallization attempts I ALWAYS use double distilled > (rectified) water. However there is, I must admit, a fume > of triple distilled Jameson around (almost always). All this is not > written in the Rigaku recipe but is very important. > > Dr Felix Frolow > > Professor of Structural Biology and Biotechnology > > Department of Molecular Microbiology > > and Biotechnology > > Tel Aviv University 69978, Israel > > > Acta Crystallographica D, co-editor > > > e-mail: mbfro...@post.tau.ac.il > > Tel: ++972 3640 8723 > > Fax: ++972 3640 9407 > Cellular: ++972 547 459 608 > > On Dec 21, 2009, at 23:02 , mjvdwo...@netscape.net wrote: > > I was going to comment that I have learned the following: "respect" does > not mean the same thing in all places in the world. Some time back I had a > protein here that I thought needed extra respect and I had learned from a > Rigaku employee how to do this - I bowed very very deeply in front of the > protein before handling it. But it still did not crystallize. So when I > complained to the Rigaku employee about the "recipe", he asked with > appropriate hesitation in his voice: are you saying that you only bowed > ONCE? > > In defense of the original poster, I think the recipe on the Rigaku web > site is entirely correct, but it does not specify how to pay proper respect. > This was self-evident to the person who wrote down the recipe, but as we all > know, what it obvious to one person, is not obvious to the next - especially > in such difficult things like respect. Good recipes are indispensable and > should be explicit about such important ingredients. > > Mark > > > -Original Message- > From: Mark J. van Raaij > To: CCP4BB@JISCMAIL.AC.UK > Sent: Mon, Dec 21, 2009 12:18 pm > Subject: Re: [ccp4bb] where I have been going wrong in crystallization? > > - I think the original poster was only calling attention to the fact that > some proteins want to be treated respectfully in order to crystallise (and > the fact that Rigaku Japan realises this). I find that indeed the case. > Other proteins, however, prefer the attitude "I don't know why I am setting > up these drops, this protein is too crappy to crystallise", i.e. a > challenge. > Lysozyme, on the other hand, even crystallises under conditions of complete > indifference. At least I find that every student in a practical course can > get nice crystals of lysozyme, and a majority of these drops have been set > up under conditions of complete indifference...maybe lysozyme is not a > protein after all, but a salt: lysozyme-chloride / LyCl7 ? > Mark > PS the detailed protocols and experiences are useful though. > > Quoting Jeffrey Wilson : > > > I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M > > NaCl method mentioned in a Hampton Research catalog and attributed to > > Enrico Stura. I see that he has also just commented on this thread. I > > found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant > > ratio, lysozyme crystallized in about 1 hour. Jumping that up to > > 150mg/ml allowed for crystallization in minutes. Hanging drop behaved > > similarly. I was using lysozyme from Sigma. > > > > Jeff > > > > Jeffrey Wilson, Ph.D. > > University of Cincinnati College of Medicine > > Molecular Genetics Department > > 231 Albert Sabin Way > > MSB 3109A > > Cincinnati, OH 45267-0524 > > (513) 558-1360 > > > > > > On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: > > > >> Dear All > >> checking out the Lysozyme crystallization methods on the web I >> liked > the Rigaku Instructions that I found: > >> (http://www.rigaku.com/protein/crystallization.html) > >> > >> "...create a drop of 3ul lysozyme solution, and 3 ul of well >> > solution, respectfully, for a total drop size of 6ul..." > >> > >> So perhaps sometimes I am just not respectful enough to deserve crystals > ? > >> > >> good wishes to all > >> regards, > >> Martyn > >> --- > >> Martyn Symmons > >> MRC-MBU Cambridge UK > >> 'Chan fhiosrach mur feòraich.' > >> Gaelic proverb - > >> Nothing asked, nothing learned. > > >
Re: [ccp4bb] where I have been going wrong in crystallization?
If "respect" is concerned, try to stop a blow of a sharp samurai sword with a sharp word. I myself have no problems whatsoever with the Rigaku recipes. And yes, I have stopped, at leas once, a blow of samurai sword with my bare hands. Since than I paint pictures with my left foot. BTW In ALL my crystallization attempts I ALWAYS use double distilled (rectified) water. However there is, I must admit, a fume of triple distilled Jameson around (almost always). All this is not written in the Rigaku recipe but is very important. Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Dec 21, 2009, at 23:02 , mjvdwo...@netscape.net wrote: > I was going to comment that I have learned the following: "respect" does not > mean the same thing in all places in the world. Some time back I had a > protein here that I thought needed extra respect and I had learned from a > Rigaku employee how to do this - I bowed very very deeply in front of the > protein before handling it. But it still did not crystallize. So when I > complained to the Rigaku employee about the "recipe", he asked with > appropriate hesitation in his voice: are you saying that you only bowed ONCE? > > In defense of the original poster, I think the recipe on the Rigaku web site > is entirely correct, but it does not specify how to pay proper respect. This > was self-evident to the person who wrote down the recipe, but as we all know, > what it obvious to one person, is not obvious to the next - especially in > such difficult things like respect. Good recipes are indispensable and should > be explicit about such important ingredients. > > Mark > > > -Original Message- > From: Mark J. van Raaij > To: CCP4BB@JISCMAIL.AC.UK > Sent: Mon, Dec 21, 2009 12:18 pm > Subject: Re: [ccp4bb] where I have been going wrong in crystallization? > > - I think the original poster was only calling attention to the fact that > some proteins want to be treated respectfully in order to crystallise (and > the fact that Rigaku Japan realises this). I find that indeed the case. > Other proteins, however, prefer the attitude "I don't know why I am setting > up these drops, this protein is too crappy to crystallise", i.e. a challenge. > Lysozyme, on the other hand, even crystallises under conditions of complete > indifference. At least I find that every student in a practical course can > get nice crystals of lysozyme, and a majority of these drops have been set up > under conditions of complete indifference...maybe lysozyme is not a protein > after all, but a salt: lysozyme-chloride / LyCl7 ? > Mark > PS the detailed protocols and experiences are useful though. > > Quoting Jeffrey Wilson : > > > I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M > > NaCl method mentioned in a Hampton Research catalog and attributed to > > Enrico Stura. I see that he has also just commented on this thread. I > > found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant > > ratio, lysozyme crystallized in about 1 hour. Jumping that up to > > 150mg/ml allowed for crystallization in minutes. Hanging drop behaved > > similarly. I was using lysozyme from Sigma. > > > > Jeff > > > > Jeffrey Wilson, Ph.D. > > University of Cincinnati College of Medicine > > Molecular Genetics Department > > 231 Albert Sabin Way > > MSB 3109A > > Cincinnati, OH 45267-0524 > > (513) 558-1360 > > > > > > On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: > > > >> Dear All > >> checking out the Lysozyme crystallization methods on the web I >> liked > >> the Rigaku Instructions that I found: > >> (http://www.rigaku.com/protein/crystallization.html) > >> > >> "...create a drop of 3ul lysozyme solution, and 3 ul of well >> solution, > >> respectfully, for a total drop size of 6ul..." > >> > >> So perhaps sometimes I am just not respectful enough to deserve crystals ? > >> > >> good wishes to all > >> regards, > >> Martyn > >> --- > >> Martyn Symmons > >> MRC-MBU Cambridge UK > >> 'Chan fhiosrach mur feòraich.' > >> Gaelic proverb - > >> Nothing asked, nothing learned.
Re: [ccp4bb] where I have been going wrong in crystallization?
I was going to comment that I have learned the following: "respect" does not mean the same thing in all places in the world. Some time back I had a protein here that I thought needed extra respect and I had learned from a Rigaku employee how to do this - I bowed very very deeply in front of the protein before handling it. But it still did not crystallize. So when I complained to the Rigaku employee about the "recipe", he asked with appropriate hesitation in his voice: are you saying that you only bowed ONCE? In defense of the original poster, I think the recipe on the Rigaku web site is entirely correct, but it does not specify how to pay proper respect. This was self-evident to the person who wrote down the recipe, but as we all know, what it obvious to one person, is not obvious to the next - especially in such difficult things like respect. Good recipes are indispensable and should be explicit about such important ingredients. Mark -Original Message- From: Mark J. van Raaij To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, Dec 21, 2009 12:18 pm Subject: Re: [ccp4bb] where I have been going wrong in crystallization? - I think the original poster was only calling attention to the fact that some proteins want to be treated respectfully in order to crystallise (and the fact that Rigaku Japan realises this). I find that indeed the case. Other proteins, however, prefer the attitude "I don't know why I am setting up these drops, this protein is too crappy to crystallise", i.e. a challenge. Lysozyme, on the other hand, even crystallises under conditions of complete indifference. At least I find that every student in a practical course can get nice crystals of lysozyme, and a majority of these drops have been set up under conditions of complete indifference...maybe lysozyme is not a protein after all, but a salt: lysozyme-chloride / LyCl7 ? Mark PS the detailed protocols and experiences are useful though. Quoting Jeffrey Wilson : > I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M > NaCl method mentioned in a Hampton Research catalog and attributed to > Enrico Stura. I see that he has also just commented on this thread. I > found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant > ratio, lysozyme crystallized in about 1 hour. Jumping that up to > 150mg/ml allowed for crystallization in minutes. Hanging drop behaved > similarly. I was using lysozyme from Sigma. > > Jeff > > Jeffrey Wilson, Ph.D. > University of Cincinnati College of Medicine > Molecular Genetics Department > 231 Albert Sabin Way > MSB 3109A > Cincinnati, OH 45267-0524 > (513) 558-1360 > > > On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: > >> Dear All >> checking out the Lysozyme crystallization methods on the web I >> liked >> the Rigaku Instructions that I found: >> (http://www.rigaku.com/protein/crystallization.html) >> >> "...create a drop of 3ul lysozyme solution, and 3 ul of well >> solution, >> respectfully, for a total drop size of 6ul..." >> >> So perhaps sometimes I am just not respectful enough to deserve crystals ? >> >> good wishes to all >> regards, >>Martyn >> --- >> Martyn Symmons >> MRC-MBU Cambridge UK >> 'Chan fhiosrach mur feòraich.' >> Gaelic proverb - >> Nothing asked, nothing learned.
Re: [ccp4bb] where I have been going wrong in crystallization?
- I think the original poster was only calling attention to the fact that some proteins want to be treated respectfully in order to crystallise (and the fact that Rigaku Japan realises this). I find that indeed the case. Other proteins, however, prefer the attitude "I don't know why I am setting up these drops, this protein is too crappy to crystallise", i.e. a challenge. Lysozyme, on the other hand, even crystallises under conditions of complete indifference. At least I find that every student in a practical course can get nice crystals of lysozyme, and a majority of these drops have been set up under conditions of complete indifference...maybe lysozyme is not a protein after all, but a salt: lysozyme-chloride / LyCl7 ? Mark PS the detailed protocols and experiences are useful though. Quoting Jeffrey Wilson : I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M NaCl method mentioned in a Hampton Research catalog and attributed to Enrico Stura. I see that he has also just commented on this thread. I found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant ratio, lysozyme crystallized in about 1 hour. Jumping that up to 150mg/ml allowed for crystallization in minutes. Hanging drop behaved similarly. I was using lysozyme from Sigma. Jeff Jeffrey Wilson, Ph.D. University of Cincinnati College of Medicine Molecular Genetics Department 231 Albert Sabin Way MSB 3109A Cincinnati, OH 45267-0524 (513) 558-1360 On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: Dear All checking out the Lysozyme crystallization methods on the web I liked the Rigaku Instructions that I found: (http://www.rigaku.com/protein/crystallization.html) "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, respectfully, for a total drop size of 6ul..." So perhaps sometimes I am just not respectful enough to deserve crystals ? good wishes to all regards, Martyn --- Martyn Symmons MRC-MBU Cambridge UK 'Chan fhiosrach mur feòraich.' Gaelic proverb - Nothing asked, nothing learned.
Re: [ccp4bb] where I have been going wrong in crystallization?
I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M NaCl method mentioned in a Hampton Research catalog and attributed to Enrico Stura. I see that he has also just commented on this thread. I found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant ratio, lysozyme crystallized in about 1 hour. Jumping that up to 150mg/ml allowed for crystallization in minutes. Hanging drop behaved similarly. I was using lysozyme from Sigma. Jeff Jeffrey Wilson, Ph.D. University of Cincinnati College of Medicine Molecular Genetics Department 231 Albert Sabin Way MSB 3109A Cincinnati, OH 45267-0524 (513) 558-1360 On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: Dear All checking out the Lysozyme crystallization methods on the web I liked the Rigaku Instructions that I found: (http://www.rigaku.com/protein/crystallization.html) "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, respectfully, for a total drop size of 6ul..." So perhaps sometimes I am just not respectful enough to deserve crystals ? good wishes to all regards, Martyn --- Martyn Symmons MRC-MBU Cambridge UK 'Chan fhiosrach mur feòraich.' Gaelic proverb - Nothing asked, nothing learned.
Re: [ccp4bb] where I have been going wrong in crystallization?
Dear Martin, The original procedure with polyethylene glycol comes from: Stura, E.A. (1998) Strategy 3: Reverse Screening. In "Crystallization of Proteins: Techniques, Strategies and Tips. A laboratory manual" (Bergfors, T., ed) International University Line. pp. 113-124. If you follow the procedure described within you should not have problems in getting crystals in 15 mins unless there is a problem with your hen egg white lysozyme. The method given on the Rigaku site proposes 25% (v/v) ethylene glycol instead of polyethylene glycol (2K-10K). This would slow down the crystallization rate. You are better off using polyethylene glycol 4K and then transfer the crystals for freezing into 15% polyethylene glycol 4K, 25% (v/v) ethylene glycol, 1M NaCl, pH 4.5 as your cryo-solution. Why wait 2 days when you can do it in 1 hour? Enrico. On Mon, 21 Dec 2009 17:12:48 +0100, MARTYN SYMMONS wrote: Dear All checking out the Lysozyme crystallization methods on the web I liked the Rigaku Instructions that I found: (http://www.rigaku.com/protein/crystallization.html) "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, respectfully, for a total drop size of 6ul..." So perhaps sometimes I am just not respectful enough to deserve crystals ? good wishes to all regards, Martyn --- Martyn Symmons MRC-MBU Cambridge UK 'Chan fhiosrach mur feòraich.' Gaelic proverb - Nothing asked, nothing learned. -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449 Lab LTMB, SIMOPRO, IBiTec-S, CEA Saclay, 91191 Gif-sur-Yvette Cedex FRANCE http://www-dsv.cea.fr/en/ibitecs/82 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] where I have been going wrong in crystallization?
Hi Martyn, this recipe tends to work for me... Lysozyme: 50 mg/ml in 0.1 M Sodium Acetate pH 4.8 Reagent: 8% w/v Sodium Chloride, 0.1 M Sodium Acetate pH 4.8 Additional Reagents: Index Reagent 8, 22, 28, 31, 34, 40, 58, 59, 69, 86, 88 Mix equal amounts of lysozyme with reagent, incubate at 4 or 22 degrees Celsius. Batch or vapor diffusion works fine. ==> Direct copy/paste from http://hamptonresearch.com/experiments.aspx <== HTH and Happy Christmas. Cheers, Dave David C. Briggs PhD University of Manchester E-mail: david.c.bri...@manchester.ac.uk Twitter: @xtaldave Skype: DocDCB 2009/12/21 MARTYN SYMMONS : > Dear All > checking out the Lysozyme crystallization methods on the web I liked the > Rigaku Instructions that I found: > (http://www.rigaku.com/protein/crystallization.html) > > "...create a drop of 3ul lysozyme solution, and 3 ul of well solution, > respectfully, for a total drop size of 6ul..." > > So perhaps sometimes I am just not respectful enough to deserve crystals ? > > good wishes to all > regards, > Martyn > --- > Martyn Symmons > MRC-MBU Cambridge UK > 'Chan fhiosrach mur feòraich.' > Gaelic proverb - > Nothing asked, nothing learned. >
