You're not the only one to have had exactly that problem with exactly that system!
A good ad for the potential utility of JDF (Journal of Dismal Failure). Phoebe ---- Original message ---- >Date: Thu, 14 May 2009 19:20:58 -0400 >From: [email protected] >Subject: Re: [ccp4bb] Crystallizing Fluorescent Molecules >To: [email protected] > >CCP4 bulletin board <[email protected]> wrote on 05/14/2009 03:42:05 >PM: > >> Dear Crystallographers, >> >> I have exactly two spherulite crystals of a protein-peptide complex which > >> have a fluorescently-labelled peptide in them, and are therefore nicely >> colorful in both the light and fluorescence microscopes, making it easier >to >> know that at least the peptide is in the crystal. However, they are not >> reproducible. Having gone through the usual list of possible variations >> which might account for the irreproducibility, I have hit the bottom of >the >> barrel. I was thinking that since the original crystals grew in utter >> darkness, undisturbed for two weeks while I was away, they were able to >> nucleate. Is it possible that light exciting the fluorophores is >detrimental >> to crystallization? Or perhaps the complete uniformity of temperature? >Even >> microseeding from one of the spherulites produced nothing (except in the >> original well.) Any brilliant suggestions welcome... >> >> Jacob Keller >> > >Hi Jacob - > >I just want to raise a warning, since a very similar situation bit me back >in grad school. I was trying to crystallize RecA with a >fluorescently-labeled oligo, and just like you, I got green-glowing >crystals, along with some glowing precipitate. I got extremely excited, >and a couple of months later had the structure done. There was no DNA in >the structure. I had crystallized unliganded RecA, and the fluorescent DNA >had precipitated all over the outside of the crystal, painting it and >making it look green! > >Are you certain that your peptide doesn't precipitate under your >crystallization conditions? I hope for your sake that my problem is not >yours... > >- Matt > > >-- >Matthew Franklin , Ph.D. >Senior Scientist, ImClone Systems, >a wholly owned subsidiary of Eli Lilly & Company >180 Varick Street, 6th floor >New York, NY 10014 >phone:(917)606-4116 fax:(212)645-2054 > > >Confidentiality Note: This e-mail, and any attachment to it, contains >privileged and confidential information intended only for the use of the >individual(s) or entity named on the e-mail. If the reader of this e-mail >is not the intended recipient, or the employee or agent responsible for >delivering it to the intended recipient, you are hereby notified that >reading it is strictly prohibited. If you have received this e-mail in >error, please immediately return it to the sender and delete it from your >system. Thank you. Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
