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Hi Tom: Glycerol is fine in crystallization trials. As you say it may increase your solubility, in your case that appears to be a good thing, for other proteins, it makes them too soluble and then one may have to increase the concentration to something more than the typical values. Glycerol is also fine during gel filtration, although because of the increased viscosity, you may have to decrease your flow rate. Hope this helps. Cheers, Alex On 3/5/2011 7:00 PM, CCP4BB automatic digest system wrote: > There are 3 messages totaling 171 lines in this issue. > > Topics of the day: > > 1. off-topic replay: glycerol in protein stock solutions (2) > 2. Philosophy and Re: [ccp4bb] I/sigmaI of>3.0 rule > > ---------------------------------------------------------------------- > > Date: Fri, 4 Mar 2011 19:25:53 -0600 > From: "Brett, Thomas"<[log in to unmask]> > Subject: off-topic replay: glycerol in protein stock solutions > > Hi guys: > I know this has been asked before, but I want to get (current) opinions and observations once again. Every once in a while, you work with a protein that needs to have a little something added to the buffer to keep it soluble. The most common trick is addition of glycerol (usually 5-10%). I'm looking for general observations on this. I was of the opinion that this was usually a bad thing to do with protein stock that you intend to cyrstallize because glycerol will coat the protein in a non-homogeneous manner and make your homogeneous protein prep heterogeneous (in a way). Or do people usually have good luck crystallizing proteins that have to be stored in some glycerol? Or is there a better additive? Also, when having to use glycerol, do you put it on your sizing columns, etc? I am concerned with putting glycerol on my columns I may never be able to completely wash it away. What are your thoughts, community? > thanks in advance, > -tom > > ------------------------------ > > Date: Fri, 4 Mar 2011 20:42:50 -0500 > From: "Van Den Berg, Bert"<[log in to unmask]> > Subject: Re: off-topic replay: glycerol in protein stock solutions > > Hi Tom, > > Adding glycerol to (crystallization) buffers is a very common practice when working with membrane proteins. Many membrane proteins have been crystallized (perhaps even the majority) with glycerol, even up to 30% v/v. So, at least for membrane proteins, there is no problem. > > Bert > > > On 3/4/11 8:25 PM, "Brett, Thomas"<[log in to unmask]> wrote: > > Hi guys: > I know this has been asked before, but I want to get (current) opinions and observations once again. Every once in a while, you work with a protein that needs to have a little something added to the buffer to keep it soluble. The most common trick is addition of glycerol (usually 5-10%). I'm looking for general observations on this. I was of the opinion that this was usually a bad thing to do with protein stock that you intend to cyrstallize because glycerol will coat the protein in a non-homogeneous manner and make your homogeneous protein prep heterogeneous (in a way). Or do people usually have good luck crystallizing proteins that have to be stored in some glycerol? Or is there a better additive? Also, when having to use glycerol, do you put it on your sizing columns, etc? I am concerned with putting glycerol on my columns I may never be able to completely wash it away. What are your thoughts, community? > thanks in advance, > -tom > > > ------------------------------ > > Date: Sat, 5 Mar 2011 08:28:47 +0000 > From: Jrh<[log in to unmask]> > Subject: Philosophy and Re: [ccp4bb] I/sigmaI of>3.0 rule > > Dear Colleagues, > Agreed! There is a wider point though which is that the 3D structure and data can form a potential for further analysis and thus the data and the structure can ideally be more than the current paper's contents. Obviously artificially high<I/ sig I> cut offs are both unfortunate for the current article and such future analyses. In chemical crystallography this potential for further analyses is widely recognised. Eg a crystal structure should have all static disorder sorted, methyl rotor groups correctly positioned etc even if not directly relevant to an article. Such rigour is the requirement for Acta Cryst C , for example, in chemical crystallography. > Best wishes, > John > > > Prof John R Helliwell DSc > > > On 4 Mar 2011, at 20:36, Roberto Battistutta<[log in to unmask]> wrote: > >> Dear Phil, >> I completely agree with you, your words seem to me the best >> "philosophical" outcome of the discussion and indicate the right >> perspective to tackle this topic. In particular you write "In the end, the >> important question as ever is "does the experimental data support the >> conclusions drawn from it?" and that will depend on local information >> about particular atoms and groups, not on global indicators". Exactly, in >> my case, all the discussion of the structures was absolutely "independent" >> from having 1.9, 2.0 or 2.1 A nominal resolution, or to cut at 1.5 or 2.0 >> or 3.0 I/sigma. This makes the unjustified (as this two-day discussion has >> clearly pointed out) "technical" critics of the reviewer even more >> upsetting. >> Ciao, >> Roberto > ------------------------------ > > End of CCP4BB Digest - 4 Mar 2011 to 5 Mar 2011 (#2011-65) > **********************************************************