| Quick navigation: | Home | Site Map || References | Biography || Copyright | Other copyright | Contact us | | |
|
|
Re: [ccp4bb] Are Calcium Citrate Crystals A Common False Positive? |
|
CCP4bb navigationCCP4bb <-- 2008 <-- April 2008 <-- 30 April 2008Subject: Re: Are Calcium Citrate Crystals A Common False Positive? From: "R {- dot -} M {- dot -} Garavito" garavito {- at -} MSU {- dot -} EDU Date: 2008-04-30 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: garavito@msu.edu **************************************************************** 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:CCP4BB@JISCMAIL.AC.UK] On Behalf > Of Jim Pflugrath > Sent: Tuesday, April 29, 2008 4:24 PM > To: CCP4BB@JISCMAIL.AC.UK > 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 > CCP4bb navigationCCP4bb <-- 2008 <-- April 2008 <-- 30 April 2008 |
| ProteinCrystallography.org: Copyright 2006-2008 by Quid United Ltd |