Joe,
What your are describing is microcrystal formation. The silkiness or
"opalescence" is very typical of microcrystalline showers.
Concerning the effect of DTT, I recall one other case where DTT-
sensitive microcrystals formed: the crystalline insecticidal toxins
produced by B. thuringiensis, which are used as specific
insecticides. Although I can't recall the details, possible
disulfide bridge formation occurred during crystal formation. Jade
Li solved its structure awhile ago (Li, Koni, and Ellar, J. Mol.
Biol. 257, 129–152, 1996) and noted:
"Crystals of the CytB protoxin were grown (Li et al., 1995) by
microdialysis in the presence of 5 mM dithiothreitol (DTT), by
reducing the pH from 8.5 to 7.2 and at the same time reducing the
concentration of a solubilizing agent, either urea from 50 mM to nil,
or ethanolamine from 8 mM to 3 mM. The presence of either urea or
ethanolamine was required, together with DTT, to control protein
aggregation."
Hope that this helps,
Michael
****************************************************************
R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
513 Biochemistry Bldg.
Michigan State University
East Lansing, MI 48824-1319
Office: (517) 355-9724 Lab: (517) 353-9125
FAX: (517) 353-9334 Email: [EMAIL PROTECTED]
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On Nov 13, 2007, at 4:09 PM, Joe wrote:
The other possibility is 400 mM imidazole in the buffer. The
precipitate looks like silk.
On 11/13/07, Bryan W. Lepore <[EMAIL PROTECTED]> wrote:
you didn't say how you know its protein - is it?
interesting though.
On Nov 13, 2007, at 4:13 PM, Joe wrote:
The precipitate does not disappear automaticly if I don't add DTT.
If it's the precipitate of imidazole, then why DTT can dissolve it?
thanks
On 11/13/07, Sanishvili, Ruslan <[EMAIL PROTECTED]> wrote:
This may not apply in your case but it is not uncommon for a
protein to
"precipitate" in a microcrystalline shower when put in cold. Once it
worms up, the crystals dissolve and the precipitate clears up. It is
easy to check under a high magnification microscope.
Cheers,
N.
Ruslan Sanishvili (Nukri), Ph.D.
GM/CA-CAT, Bld. 436, D007
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439
Tel: (630)252-0665
Fax: (630)252-0667
[EMAIL PROTECTED]
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Joe
Sent: Tuesday, November 13, 2007 2:30 PM
To: [email protected]
Subject: [ccp4bb] DTT sensitive?
Hi there,
I see enormous precipitate of my receptor protein when I take it
out of
freezer. But all the precipitate dissolved quickly after I added
1mM DTT
to the solution. Does this mean that some surface Cys are causing
problem? Or why is the protein so sensitive to DTT?
Anybody experienced this kind?
Any advice is appreciated.
-Joe
