Agarose gels for xtal growth

Mark van der Woerd woerd at orion.cmc.uab.edu
Sat Dec 11 17:14:00 EST 1993


In article <1993Dec9.223638.20145 at usage.csd.unsw.OZ.AU>, sjt at newt.phys.unsw.edu.au (Sarah Tilley) writes...
> 
>-- 
>Hi,
> 
>I thought I would try my hand at growing xtals in agarose gels, and I am 
>trying to follow the procedure in Ducruix and Giege's book, "Crystallization 
>of Nucleic Acids and Proteins". They suggest a method for a 36 deg C gelling 
>point agarose, but there ar

[e ... no i]

>dea what gels are routinely used. Any recommendations? I don't want to work 
>with silica gels, as I want to use conditions previously used for hanging 
>drops and I intend to grow xtals in gels using the same method. Any opinions 
>on using gels? Anything I should be particularly careful about? H
>
>Thanks in advance
> 
>**
>Sarah Tilley
>sjt at newt.phys.unsw.edu.au

Well, we have grown crystals in gels. See, we think that one of the ways that 
protein crystals may become of inferior quality, sometimes, is convection in 
the solution. Gels prevent convection. There is another advantage for you, 
possibly. It is a pain to remove gel from crystals, once you have them. Instead 
of mounting crystals, you cut them out of pudding. :-) This pudding, however, 
at the same time protects your crystals. So, I'd think that you might find that 
your crystals splinter less (once you have some, of course).

We used both agarose and silica. Both work. The problem is usually to get 
sufficiently big crystals, as they grow slower and/or to a smaller final size 
in the case of gels. So far (only one experiment done, I know of) we found 
silica gel giving smaller crystals than agarose. The trick is, you must have a 
very low percentage of agarose. You want to give the crystals support, but you 
don't want to totally deprive the crystallites of protein because the gel-maze 
is too dense.
Also, you must be careful with temperature and always want to make sure that 
your protein is not exposed to high temperatures (> 40 C). I think I have seen 
recipes that either call for dissolving protein in the warm gel-solution before 
solidification, or for soaking proteins into the gel etc.

When you want to get some experience with gels, start with lysozyme. This is 
easy and cheap. Then try whatever it is you are after. Note that many proteins
will NOT crystallize in gels under the same conditions that they DO crystallize 
with hanging drop method. I think I vaguely recall a statement by Dr. McPherson
(the crystallization expert, author of book) that he could only crystallize a 
small percentage (25% ?) of proteins tried in gels, for which the proper 
conditions in hanging drops are known. Conditions may be very different !

Right now I cannot give you any recipes, as I have not crystallized anything 
myself in gels... will the above help ?

Mark van der Woerd			Internet: woerd at io.cmc.uab.edu
Univ. of Alabama at Birmingham		Bitnet: CMC0001 at UABDPO
Dep. of Chemistry
===============
Handy Guide to Modern Science: 
   1.  If it's green or it wiggles, it's biology.
   2.  If it stinks, it's chemistry.
   3.  If it doesn't work, it's physics.
===============




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