Donna M Zukaitis-Falcone
v118lsca at ubvms.cc.buffalo.edu
Mon Nov 8 10:45:00 EST 1993
In article <pascal-061193173955 at 220.127.116.11>, pascal at seine.ceng.cea.fr (Pascal MARTINEZ) writes...
> OK I'd like to have some advices : I'd like to obtain a xtal from a
>protein of about 92.000. I need some very plain and (I'm afraid) basical
>recomandations to do so. Our main problem is that this protein is sometimes
>tyrosinated and glycosylated depending on the way we express it. Are these
>groups rumoured or not to interfere with the formation of a xtal ?
> PS: any convenient and practical handbook for protein xtallization ?
>DBMS Laboratoire du Cytosquelette
>Unite INSERM 366
>38041 Grenoble cedex
There is an excellent review article on macromolecule crystallization by
Alexander McPherson(European Journal of Biochemistry 189 1-23 1990) which you
may find useful. Also, there is an excellent book, by Alexander McPherson on
the same subject. Both are excellent resources for your problem.
I have worked with a glycoprotein 40 kDin mass,with 20% asp linked
glycans, with some success. I don't know anything about tyrosinated proteins
and xtalization. I generally work with the protein supersaturated in an
ammonium sulfate solution that is buffered to the pH range I wish to work in.
For example, a Na Acetate can be used--by pH adjusting NaOH+glacial acetic acid
to the range of interest. Then add Ammonium Sulfate (AS)in varying degrees
ie. AS 10% + Buffer, AS 20% + Buffer , AS 40% + buffer. to this add your
protein in excess so that it is supersaturated. Finally use a hanging drop
set up for crystallization with AS as a dessicant at twice the Conc. as in your
This is how I do my Xtalizations for example..............
Get a Linbro plate, and siliconized slide coverplates, and vacuum grease. use
as an example....
your protein in a buffered solution with 40%AS....add a 10 microliter droplet
to the center of a siliconized cover slide. To a well in the linbro plate add
1 ml of 80% AS. Use vacuum grease to generously coat the lip of the well. Put
the cover plate over the well with the drop hanging down( dont let the drop
comtact the dessicant of course). press down the cverplate to get a tight seal.
repeat the process varying AS% and Protein saturation. Leave the set upo in a
cool dry place and PRAY.
You'll find Xtalization is more a black art than a science. Good luck and please
look up those references for details!!!!!!!!!!
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