his tag protein storage

*.* Troianovsk_s at MSDISK.WUSTL.EDU
Wed Feb 1 14:56:49 EST 1995


>Date:   24-JAN-1995 15:08
>From: sl16s at cc.usu.edu (NAME RAVI PRAKASH .D)
>Description: his tag protein storage
>
>I am trying to purify His tag fusion protein in pET vectors.
>I would be great full if someone can tell me how I can store
>the protein for long term. If anyone has any expertise in this 
>matter I would be happy toar from you.
>thank you vey much.
> 
   Hi, there. You forgot to mention, at which conditions
(native/denaturated) you purify your protein.

   Since sprig last year, I have my proteins samples stored at -85.
Routinely I`ve freeze and thaw one tube and for short term storage used a
-20 freezer. As long as 6 month late I compared the samples in SDS-PAGE, and
in ligand blot experiments. The gel after SDS-PAGE I stained by silver
nitrate. There was no difference between the samples in both experiments.
However, I`d recommend to aliquot the samples and store them at -85 (read
-70, in our department all deep freezers adjusted to -85), and for routine
experiments store sample at -20.
The samples mentioned above were in elution buffer from Ni-NTA resin (8 M
Urea; 0.1 M NaH2PO4; 10 mM Tris; 10 mM b-MEt; 0.1 mM PMSF, 1 mM Benz*HCl; pH
4.5).
   In case you purify the protein at native conditions, I`d recommend
following extra addition to the basic buffer: 2-5 mM b-Met; 40-30% Glycerol;
1-0.2% NP-40; 5-10 mM MgCl2; the NaCl and KCl should be at physiological
concentrations (as in PBS).
-----------
           Nikolai. 

Child says: "If All Fails Carefully Read the Manual"
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