Marker Gene Monthly Newsletter   

December, 2003

Volume 3, Number 12

© Copyright MGT, Inc., 2007.  Published by Marker Gene Technologies, Inc., The University of Oregon Riverfront Research Park, 1850 Millrace Drive, Eugene, Oregon 97403-1992 USA.  All rights reserved.  For information on the use or copying of the material contained in this document, please contact us at techservice@markergene.com.  Please see below for subscription information and updates.  This newsletter is labeled as an ADVERTISEMENT in accordance with the CAN-SPAM act of 2003, S.877 Public Law: 108-187.

"Fluorobodies" combine GFP with Antibody binding.

By substitution of several exposed loops on the GFP backbone with antibody regions that specifically bind to targets, Andrew Bradbury and colleagues at Los Alamos National Laboratory have generated a “fluorobody” that retains the fluorescent characteristics of GFP while also being able to function as an antibody. The net result is a simplification of the detection process and potential improvement in detection sensitivity.  They used of a novel form of GFP (superfolder GFP) that is more stable than traditional GFP, and is able to tolerate genetic fusion to poorly folding proteins and remain fluorescent. A second modification involves the use of CDRs from antibodies as diversity elements, rather than random peptides encoded by oligonucleotides. These fluorobodies bind their targets specifically as shown by band shift assays, ELISA’s and crude microarrays and hold tremendous potential in the diagnostic arena, as well as in proteomics, where they are likely to become the binding ligand of choice.  For more information about these new reagents, see the references below:

• Zeytun, A.; Jeromin, A.; Scalettar, B. A.; Waldo, G. S.; Bradbury, A. R., "Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies," (2003) Nature Biotechnology, 21: 1473-1479 (2003)
• Bradbury, A., Velappan, N., Verzillo, V., Ovecka, M., Chasteen, L., Sblattero, D., Marzari, R., Lou, J., Siegel, R., Pavlik, P., “Antibodies in proteomics II: screening, high-throughput characterization and downstream applications” (2003) Trends in biotechnology. 21(7): 312-318.
• Bradbury, A., Velappan, N., Verzillo, V., Ovecka, M., Chasteen, L., Sblattero, D., Marzari, R., Lou, J., Siegel, R., Pavlik, P., “Antibodies in proteomics I: generating antibodies”, (2003)Trends in biotechnology. 21(6): 275-282.
• Kim, I.S., J.H. Shim, Y.T. Suh, K.Y.F. Yau, J.C. Hall, J.T. Trevors & H. Lee (2002) Green fluorescent protein-labeled recombinant fluorobody for detecting the picloram herbicide.  Bioscience, Biotechnology and Biochemistry 66:1148-1151.
  

The CCPGCC Tetracysteine Reporter Gene.

Roger Tsien’s lab has introduced a series of non-fluorescent membrane-permeant biarsenical dyes (FlAsH, ReAsH, etc.) that form specific fluorescent covalent complexes with an intracellular protein to which a short tetracysteine-containing peptide (CCPGCC) has been genetically fused.  The small size of the biarsenical dyes is advantageous in those cases where a bulky GFP or Aequorea fluorescent protein fusions disrupt normal protein function.  The arsenic compound is bound with ethanedithiol groups to minimize the toxicity of any unbound biarsenical-dye.  Sequential labeling with different biarsenical dyes can indicate the age of intracellular protein molecules.  The tetracysteine motifs can be labeled rapidly and saturably with one colour of a membrane-permeant biarsenical dye, such as a green FlAsH (a fluorescein derivative) then any free dye is washed out and the live cells allowed to synthesize fresh unlabelled copies of the same tagged protein.  Next, exposure of the newly synthesized proteins to a second biarsenical dye of a different color, such as red ReAsH, labels only the newly synthesized copies. This approach was used to study the life cycle of connexin-43 as it was trafficked into and out of gap junctions (see the figure at right).  For more information about these new reporter gene techniques, see the references below:

• Griffin, B.A., Adams, S.R., Jones, J., Tsien, R.Y. (2000) “Fluorescent labeling of recombinant proteins in living cells with FlAsH 2001”. Methods in Enzymology. 327: 565-578.
• http://health.ucsd.edu/news/2002/04_18_Ellisman.html

New Chemiluminescent LacZ ß-Galactosidase Detection Kit.

Marker Gene introduced a new chemiluminescent assay kit for detection of b-galactosidase at the American Society for Cell Biology Meeting in San Francisco last week.  The E. coli lacZ gene codes for an active subunit of ß-galactosidase in live cells.  Since this enzyme is generally absent in normal mammalian, yeast, some bacterial and even plant cells, it can be used as a fusion marker, and detected at very low levels.  In addition, its wide substrate specificity allows  monitoring of lacZ expression (and therefore co-expressed genes or promoter efficiency) of as few as 5 copies of the ß-galactosidase protein. Although chromogenic assays of ß-galactosidase activity (i.e. X-Gal) are useful, the recent application of chemiluminescent 1,2 dioxetane substrates, which emit visible light upon enzyme catalysis, provide rapid results with very low background and high intensity signal.  Our results, presented at the meeting, indicate that this assay may be as much as 30X more sensitive than conventional assay techniques, in live cells.  An enhancing solution is also provided with this kit to increase light production efficiency. The Chemiluminescent lacZ ß-Galactosidase Detection Kit (M0855) provides all the necessary reagents, buffers, substrate, and a detailed protocol for sensitive and quantitative lacZ ß-galactosidase activity assay.  Please contact our technical services department for further information, and see the references below for more details.

• “New β-Galactosidase Chemiluminescent Live Cell Assay System.” L. Tsai, B. Giri, H. R. Toben, J. J. Naleway, (2003) Mol. Biol. Cell 14: 468a.
• Chemiluminescent 1,2-dioxetanes. Brij P. Giri, PCT/US99/20590.
• Novel stabilized formulations for chemiluminescent assays. Brij P Giri, PCT/US01/02779.
• Single molecule detection of Alkaline Phosphatase enzyme using enhanced chemiluminescent from 1,2-dioxetanes and water-soluble, water-insoluble or partially water-soluble polymers. Brij P Giri, US Patent Application, 2002/0013250.

New Dehalogenase Reporter Gene.

Dr. Georgyi Los and collaborators at Promega Biosciences introduced a new reporter gene system for monitoring cloned gene expression in mammalian cells at the 43^rd Annual American Society for Cell Biology Meeting in San Francisco last week.  This new system utilizes a mutant Rhodococcus rhodochrous haloalkane dehalogenase (DhaA) enzyme fused to a gene of interest for monitoring gene expression in mammalian cell lines.  Combined with several fluorescent dyes that have a halo-alkyl group attached (TAMRA, ROX, FAM etc.), the new fusion genes can be used to monitor gene expression events intracellularly.  In this way a DhaA-b-arrestin2 fusion protein was monitored in CHO-K1 cells.  For more information about this new reporter gene and the assay systems, see the reference below or visit the Promega website at www.promega.com .

• G. V. Los, C. Zimprich, M. McDougall, N. Karassina, R. Learish, D. Klaubert, K. Wood, B. Bulleit “Targeting of Small Molecule Reporters Within Living Cells”  43^rd Annual ASCB Meeting, San Francisco, Dec. 2003, Presentation Number: 2612
  Poster Board Number: B343.
  
  

 News from the American Society for Cell Biology Meeting in San Francisco.

Marker Gene presented several papers at the 43^rd Annual Cell Biology Meeting in San Francisco, December 13-17, 2003 at the Moscone Convention Center.  These include the new chemiluminescent assay for lacZ (see above), as well as new luciferase and GUS systems for cell regulation.  For more information, please visit the Society’s web site at www.ascb.org. 

•   “β-Glucuronidase Mediating Drug Delivery System in LFY::GUS Arabidopsis thaliana.” L. Tsai, M. Braden, T. Neff, J. J. Naleway, , (2003) Mol. Biol. Cell 14: 393a.
•  “New β-Galactosidase Chemiluminescent Live Cell Assay System.” L. Tsai, B. Giri, H. R. Toben, J. J. Naleway, (2003) Mol. Biol. Cell 14: 468a.
•  “New Luciferin Analogs for Monitoring Cloned Luciferase Activity.” L. Tsai, A. P. Guzilowski, J. J. Naleway, L277 Presented at the 43^rd Annual Cell Biology Meeting in San Francisco, Dec.,2003.
  
  

2004-2005 Catalog Will Be Available Soon.

The 2004-2005 edition of the Marker Gene catalog is in production.  Many new products and kits, additional literature references, data and protocols will be included, as well as new information about our old products.  Be sure to add your name to our mailing list.  Please visit our Web site and fill out our Customer Information Form, or e-mail us at techservice@markergene.com and we will have a copy sent out to you.
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Marker Gene strives to offer our customers products of the highest quality and at the best possible prices.  Our years of experience allow us to provide timely products for less cost to you.  See our latest Price Comparison Chart that compares our prices with those from several alternate sources, to see if you can save money by switching to Marker Gene (http://www.markergene.com/crossref.htm).  Or visit our website at www.markergene.com and click on the link “COMPARE”.  We think you will appreciate our efforts to keep costs low and maintain excellent quality of our products for your research.  For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com.  We will be happy to send you more about our products and their specifications.

CONTRACT  RESEARCH@markergene.com

Marker Gene Technologies, Inc. has the expertise to perform contract research with you on your project. We have worked with many biotechnology and pharmaceutical companies on successful, proprietary and patented projects.

Contract Research and Development Capabilities in the following areas:

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays – High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
• Confidentiality, help in patent preparation and filings.

Contact us by telephone at (888) 218-4062 or (541) 342-3760 or FAX us at (541) 342-1960 or you can write to us at  Contract Research, Marker Gene Technologies, Inc., 1850 Millrace Drive, Eugene, Oregon 97403-1992 or contact us by e-mail at: techservice@markergene.com

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