Calcium citrate does have a relatively low solubility (~15 mM in the
cold), and its solubility decreases as the temperature goes up.
Thus, getting calcium citrate crystals is a possibility if both are
at 200 mM. However, you can get sodium citrate up to ~1.4 M at
least, depending on the pH.
If they are harvestable crystals, just drop them into a low ionic
strength buffered solution containing 0.2%-2% glutaraldehyde.
Protein crystals will quickly be fixed quickly (faster than they
dissolve) into a light golden, gelatinous lump. Sometimes they retain
a crystal-like shape, other times they leave just a rubbery drop. In
contrast, salt crystals should dissolve over time and should not be
colored.
You could also add a small drop of 2% glutaraldehyde to your protein
drop. Protein crystals then turn a light golden color. Crystals
fixed like this can then be put into a low ionic strength solution
where salt crystals should dissolve. You can easily transfer
glutaraldehyde into a protein drop by vapor diffusion by adding
glutaraldehyde to the reservoir to make it 2-3%. Glutaraldehyde is
quite volitile. If you can smell it, it is fixing your olfactory
cells and corneas. Using solutions of glutaraldehyde less than 1% is
generally safe.
The only caveat to using this method is that there should be no free
amines around other than on the protein (i.e., no ethanolamine or
Tris buffer, no ammonium ions, etc.). I have never found a protein
crystal I couldn't fix; I always end up with a gelatinous lump, at
least. But there probably is an exception or two. I prefer this
method over Izit and other dye methods.
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]
****************************************************************
On Apr 29, 2008, at 9:42 PM, Dunten, Pete W. wrote:
And how will you know if they are the calcium citrate xtals Sam
asked about, and not
sodium citrate xtals? Sodium citrate in the Hampton Crystal Screen
condition will
crystallize out at 4 degrees. It's solubiility is pH and
temperature dependent.
You'll need to go to your friendly neighborhood synchrotron and
either look at the
x-ray fluorescence emission spectrum to see if calcium is there, or
collect a dataset
and solve the structure. (Setting myself up here for a reply from
Bruker about how
easy the latter would be).
Pete
-----Original Message-----
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf
Of Jim Pflugrath
Sent: Tuesday, April 29, 2008 4:24 PM
To: [email protected]
Subject: Re: [ccp4bb] Are Calcium Citrate Crystals A Common False
Positive?
That's an easy hypothesis to test. Simply set up your drops with
the same conditions except without protein and see if you get
crystals. Please let us know the results. Thanks!
Jim
On Tue, 29 Apr 2008, Sam Stephenson wrote:
Are calcium citrate crystals a common false positive in trays with up
to 200mM of each? There is absolutely no phosphate in the trays so
I'm almost positive they're not calcium phosphate. Cheers, Sam
