Advices needed

Laurent Maveyraud maveyrau at ecstasy.NoSubdomain.NoDomain
Wed Nov 10 14:04:24 EST 1993


You might be interested in reading Methods in enzymology vol 114, the second section is about protein crystallization.

> 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.  

When using ammonium sulfate as the precipitant, it is more usefull to buffered the well solution than the drop, since ammonium is volatile, the drop will reach th pH of the well. Moreover, AS is not the only precipitant agent you can use. The more common are:
	Poly ethylene glycol (MW = 6000 is the more used I think)
	Methyl-2,4-pentanediol
 both are non volatile, in opposition to AS.

For the protocol to be used, I prefer mixing protein solution and precipitant just in the drop, before sealing the system over a concentrated well. At time = 0, your protein is undersaturated, but while vapour diffusion occur, the portein concentration raise slowly. If you mix too much protein in order to be sursaturated at once, you are more lucky to have amorphous precipitate, because the kinetic of concentration is too fast. Be aware that the kinetic of equilibrium might be very important for crystalliz
ation.

>repeat the process varying AS% and Protein saturation. Leave the set upo in a
>cool dry place and PRAY.  

I think it is more important to have a really stable temperature, than to be at cool temperature. A lot of protein crystallize at 16 degre or 20 degre, not only at 4 degre. Of course, if your protein is temperature sensitive, make your crystallization at cold temperature/

>You'll find Xtalization is more a black art than a science. Good luck and >please

A lot of people are trying to chance this, but it seems that it is still true for the moment. Let hope it will change soon....



By the way, Philippe, is it possible to know what is your protein ?


Good luck


Laurent Maveyraud
Groupe de Cristallographie Biologique du LPTF, CNRS
29 rue J. Marvig
31055 Toulouse
France
phone : (33) 62.25.79.63
fax   : (33) 62.25.79.60
email : maveyrau at cemes.fr




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