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"<[email protected]>
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
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Date: Fri, 4 Mar 2011 20:42:50 -0500
From: "Van Den Berg, Bert"<[email protected]>
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"<[email protected]> 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<[email protected]>
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<[email protected]>
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
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End of CCP4BB Digest - 4 Mar 2011 to 5 Mar 2011 (#2011-65)
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