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Thanks George,
These references will be helpful.  I probably need to learn more about
the Fluorescein MESF method; my concern is that, under standardized
conditions, the quantum yield of various clones that people commonly use
may vary somewhat.  It would be nice to be able to demonstrate a
quantitative positive control to users in some circumstances.  New
records for "the dimmest expression in the world" seem to be set at
increasing frequency as time goes on, it would be nice if I could give
investigators some idea of where the detection limits are for various
instruments/optics configurations.
-Karl

Mcnamara, George wrote:

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Hi Karl,
>
>Rabut et al 2004 Nat Cell Biol
>(http://www.embl-heidelberg.de/ExternalInfo/ellenberg/homepage/pdfs/Rabut200
>4,NCB6,1114.pdf) obtained 120 GFP rotavirus like particles from one of the
>authors (Cohen) of Dundr et al 2002 (pubmed abstract
>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Ab
>stract&list_uids=12490157
><http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=A
>bstract&list_uids=12490157> ), who is also an author on the original paper
>that there are 120 GFPs in the VLP: A. Charpilienne et al 2001 (JBC - free
>text at http://www.jbc.org/cgi/content/full/276/31/29361)
><http://www.jbc.org/cgi/content/full/276/31/29361)> .
>
>The Dundr et al paper also references a couple of papers using His-tagged
>GFP bound to Ni-chelate beads and similar approaches.
>
>In the absence of photobleaching (i.e. no oxygen) why not standardized
>against fluorescein MESF?
>
>
>George
>
>                -----Original Message-----
>                From:   Karl Garsha [mailto:[log in to unmask]]
>                Sent:   Tuesday, May 03, 2005 7:56 AM
>                To:     [log in to unmask]
>                Subject:        GFP standards or purified GFP?
>
>                Search the CONFOCAL archive at
>                http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>                While I'm thinking of it, does anyone know of a source of
>calibration
>                standards for work with fluorescent proteins, or
>alternatively a source
>                of purified GFP for creating such a set of standards?  With
>enough
>                motivation I'm sure one could grow up a bunch of cells,
>solublize and
>                affinity purify the GFP, dialize it to high concentration,
>assay the
>                concentration, and then make a serial dilution for dot-blot
>standards,
>                but this would be a huge amount of work.  It would be nice
>to be able to
>                buy purified GFP, CFP, YFP etc.  With all the attention on
>                high-throughput instrumentation and quantitative assays, I
>would think
>                that someone would be selling calibration standards for
>fluorescent
>                proteins.  Thanks in advance for any suggestions.
>                Best,
>                Karl
>
>                --
>                Karl Garsha
>                Light Microscopy Specialist
>                Imaging Technology Group
>                Beckman Institute for Advanced Science and Technology
>                University of Illinois at Urbana-Champaign
>                405 North Mathews Avenue
>                Urbana, IL 61801
>                Office: B650J
>                Phone: 217.244.6292
>                Fax: 217.244.6219
>                Mobile: 217.390.1874
>                www.itg.uiuc.edu
>
>

--
Karl Garsha
Light Microscopy Specialist
Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 North Mathews Avenue
Urbana, IL 61801
Office: B650J
Phone: 217.244.6292
Fax: 217.244.6219
Mobile: 217.390.1874
www.itg.uiuc.edu