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 <mbfro...@post.tau.ac.il>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 <mark.vanra...@usc.es> > 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 <wil...@ucmail.uc.edu>: > > > 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. > > >
