Marker Gene Monthly Newsletter   

Month, 2007

Volume 5, Number 7

© 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.

Fluorescent X-Gal Substrates.

A popular chromogenic substrate for detection of b-galactosidase activity in live cells is 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-gal).  It allows quick analysis of lacZ activity in fixed cells by forming an insoluble blue indigo-type dye at the location of enzyme activity.  Recently, a new fluorescent precipitating  substrate, Fluor-X-Gal, has been developed by Dr. Helen Blau and co-workers at the Department of Molecular Pharmacology, Stanford University Medical Center to provide increased detection of b-galactosidase in live cells and to permit simultaneous microscopic visualization of other fluorescent markers. Cells can be labeled with Fluor-X-Gal for fluorescent histochemical detection of b-galactosidase by culture on sterile glass coverslips (Becton Dickinson), fixed in 4% paraformaldehyde in PBS and rinsed twice with PBS (where they can be stored at 4°C until staining is performed). If cells are to be labeled with an antibody as well as Fluor X-Gal, cells should be blocked for 30 min in PBS + 10% equine serum, incubated for 2 hrs in primary antibody, rinsed for 10 min, 4 times in blocking buffer, and then incubated 1 hr in biotinylated secondary antibody.  After washing again 4 times with blocking buffer, the primary antibody can be visualized by incubating with a fluorescent-labeled streptavidin (diluted 1:100) for 1 hr., followed by washing 2 times in blocking buffer and 2 times in PBS. All such immunolabeling steps should be performed at 4^oC. The Fluor-X-Gal substrate can be used for staining fixed cells by incubating for 60-90 minutes with a 0.4 mM solution at 37^oC, then rinsing in 25 ml PBS for 30 min at room temperature prior to visualization.  Coverslips are mounted in PBS (no glycerol-based anti-fade solution is necessary) and can also be sealed (with clear nail polish) for later observation.  Fluor X-gal staining can be viewed with either fluorescein (FITC) or rhodamine (TRITC) filter sets of an epifluorescence microscope.  Marker Gene Technologies is currently in negotiations  to bring you this new useful fluorescent substrate to you.  Please visit our website or contact us at techservice@markergene.com for more information on availability.  Additional information is available in the references below.

• Rossi F., Charlton C.A., Blau H.M.  (1997) “Monitoring protein-protein interactions in intact eukaryotic cells by b-galactosidase complementation” Proc. Natl. Acad. Sci. USA 94: 8405-8410.
• Mohler W.A., Blau H.M. (1996) “Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.” Proc. Natl. Acad. Sci. USA 93(22): 12423-12427.
• Charlton, C.C., Mohler, W.A., Radice, G.L., Hynes, R.O., Blau, H.M. (1997) “Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture”, J. Cell Biol. 138(2): 331-336. 
• Corcoran, R.B.,  Scott, M.P., (2001)  “A mouse model for medulloblastoma and basal cell nevus syndrome” J. Neuro-Oncology 53: 307–318.
  

New Dual-Function Fluorescent Glutathione Transferase Assay Kit.

Glutathione (GSH), is a tripeptide (g-glutamyl-cysteinyl-glycine) that represents the major free thiol in most living cells.  It is involved in many biological processes including detoxification of xenobiotics, removal of hydroperoxides, and maintenance of the oxidation state of protein sulfhydryls.  It is the key antioxidant present in animal tissues, and diminished glutathione levels have been observed in the early stages of apoptosis.  Older methods for determining glutathione utilize 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB or Ellman’s reagent) in an oxidation or oxidation-reduction scheme.  But these methods have been largely replaced by a widely used fluorescent method for determining both GSH and glutathione transferase enzyme (GST) activity in living cells.  Marker Gene now carries  the very useful Live Cell Glutathione Transferase Activity Kit (M1045) that makes it possible to measure both glutathione levels (GSH) as well as enzyme activity in live cells.

By simply adding monochlorobimane to cell culture medium and allowing intracellular glutathione S-transferases to form GSH-mCB complexes, levels of GSH can be measured fluorometrically.  The absorption and emission maxima of the formed complex are 380nm and 461nm, respectively.  Unlike other bimanes such as monobromobimane, monochlorobimane appears to form an adduct exclusively with GSH.  The membrane-permeant monochlorobimane has been used to measure both GSH and GST levels in live cells, tissue homogenates, and in flow cytometry systems.  Several laboratories have also used this approach to measure the GSH content in cytosolic fractions of liver and intact tissues.  Our new kit contains enough reagents for up to 100 labelings, including lysis buffer, solvents, and a detailed protocol for use.  Please see our website for more information about these techniques, and look for new products from Marker Gene in this area.  Additional information is available in the references listed below.

• Fernandez-Checa J.C., Kaplowitz N. (1990) “The use of monochlorobimane to determine hepatic GSH levels and synthesis.”  Anal. Biochem. 190: 212–219.
• Kamencic H., Lyon A., Paterson P.G., Juurlink B.H.J. (2000) “Monochlorobimane Fluorometric Method to Measure Tissue Glutathione.” Anal. Biochem. 286: 35–37.
• Reichelt W., Stabel-Burow J., Pannicke T., Weichert H., Heinemann U. (1997) “The glutathione level of retinal Müller glial cells is dependent on the high-affinity sodium-dependent uptake of glutamate.” Neuroscience 77: 1213–1224.
• Devesa A., Oconnor J.E., Garcia C., Puertes I.R., Vina J.R. (1993) “Glutathione metabolism in primary astrocyte cultures: flow cytometric evidence of heterogeneous distribution of GSH content.” Brain Res. 618: 181–189.
• Kannan R., Tang D., Mackic J.B., Zlokovic B.V., Fernandez-Checa J.C. (1993) “A simple technique to determine glutathione (GSH) levels and synthesis in ocular tissues as GSH-bimane adduct: application to normal and galactosemic guinea-pigs.” Exp. Eye Res. 56: 45–50.
• Butcher E.C., Scollay R.G., Weissman I.L. (1980) J Immunol. Methods 37(2): 109-121.

Endothelial Lipase Assays.

The triglyceride lipase gene family plays a central role in dietary fat absorption, energy homeostasis, and plasma lipoprotein metabolism. Its members include pancreatic lipase, lipoprotein lipase (LPL), and hepatic lipase (HL). The latter two enzymes function in the plasma compartment and are critical to the metabolism of lipids carried on plasma lipoproteins.   Like LPL and HL, endothelial lipases (EL) also function in the plasma compartment, and initial reports have shown that overexpression of EL in mice profoundly alters plasma lipoprotein levels.  The presence of EL on the endothelium lining of blood vessels in numerous organs, its presence in macrophages, and the report that its transcription is induced by inflammatory signals also raises the possibility that this enzyme may play a role in the response of the vascular wall to injury and therefore a direct role in the formation of atherosclerotic lesions.  Analysis of endothelial lipases can be made using our Fluorescent Lipase Assay Kit (M0612).  The fluorescent fatty acid substrate provided in our kit, 1,2-dioleoyl-3-pyrenyldecanoyl-rac-glycerol (M0258), contains a triglyceride base, making it a useful substrate for measuring endothelial lipase activity.  Although data has suggested that lipoprotein lipases and hepatic lipases have higher activity for triglycerides, endothelial lipase still has about 65% activity for triglycerides when compared to phospholipids (McCoy, et al., 2002).  Our Fluorescent Lipase Assay Kit can be used both in vivo and in vitro provided that artificial lipid bilayer, micelles, or vesicles are added when using a purified enzyme system to solubilize both the substrate and enzyme.  Please see our website for more information about these techniques.  Additional information is available in the references listed below. 

• McCoy M.G., Sun G.S., Marchadier D., Maugeais C., Glick J.M., Rader D.J. (2002) "Characterization of the lipolytic activity of endothelial lipase." Journal of Lipid Research 43: 921-929.
• Broedl U.C., Jin W., Fuki I.V., and others. (2004) "Structural basis of endothelial lipase tropism for HDL." FASEB J 18(15): 1891-1893.
• Choi S.Y., Hirata K., Ishida T., Quertermous T., Cooper A.D. (2002) "Endothelial lipase :a new lipase on the block." Journal of Lipid Research  43: 1763-1769.
• Duong M., Psaltis M., Rader D.J., Marchadier D., Barter P.J., Rye K.A. (2003) "Evidence that hepatic lipase and endothelial lipase have different substrate specificities for high-density lipoprotein phospholipids." Biochemistry 42(46): 13778-85.
• Jaye M., Lynch K.J., Krawiec J., Marchadier D., Maugeais C., Doan K., South V., Amin D., Perrone M., Rader D.J.  (1999) "A novel endothelial-derived lipase that modulates HDL metabolism." Nature Genetics 21: 424 - 428.
• Hirata K., Ishida T., Matsushita H., Tsao P.S., Quertermous T. (2000) “Regulated expression of endothelial cell-derived lipase.” Biochem. Biophys. Res. Commun. 272: 90–93.
• Nilsson-Ehle P., Schotz M.C. (1976) “A stable, radioactive substrate emulsion for assay of lipoprotein lipase.” J. Lipid Res. 17: 536–541.

Environment Sensitive Fluorescent Lipid - C12-NBD.

Nitrobenzoxadiazole (NBD) is a highly environment sensitive fluorophore, becoming highly fluorescent in aprotic solvents while remaining almost nonfluorescent in aqueous environments.  Now available at Marker Gene is the 12-carbon fatty acid analog of NBD, 12-(7-Nitrobenzofurazan-4-ylamino)dodecanoic acid (C12-NBD) (M1080), a useful fluorescent probe for fatty acid and sterol carrier protein binding sites.  C12-NBD can be applied to sensing the lipid-water interface region of membranes, becoming buried in the hydrophobic interior of the lipid bilayer upon contact, making it useful for sensing lipid fluidity and structural disturbances by proteins or other additives.   C12-NBD can also be used in the synthesis numerous fluorescent phospholipids that are valuable for analysis of lipid environment or as substrates for various lipases.  Please visit our website or see the references below for more information about this new fluorescent lipid.

• Schroeder F, Myers-Payne SC, Billheimer JT, Wood WG. (1995) "Probing the ligand binding sites of fatty acid and sterol carrier proteins: effects of ethanol." Biochemistry 34(37): 11919-27.
• Tocanne JF, Dupou-Cezanne L, Lopez A. (1994) "Lateral diffusion of lipids in model and natural membranes." Prog Lipid Res 33: 203-237.
• Dao HN, McIntyre JC, Sleight RG. (1991) "Large-scale preparation of asymmetrically labeled fluorescent lipid vesicles." Anal Biochem 196: 46-53.

Now Order Marker Gene Products from VWR and Fisher Scientific!

Marker Gene has established distribution agreements with VWR Scientific Products and Fisher Scientific Products for your convenience.  Now you can order directly from these vendors using your existing account, credit card, or contract.  Many of Marker Gene’s products are already up on their websites, and you can search for our products using our product numbers.  The following information is also helpful when placing your order:

• VWR: our vendor number is 8070435, call 1-800-932-5000 and ask your account representative for assistance or contact Chris Townsley (1-856-241-7883 or Denise (1-805-313-1322) for more assistance with our products.
• Fisher Scientific: our vendor number is VN082180, call 1-800-766-7000 and ask your account representative for assistance or contact Matt Brinker (1-412-490-8006) or Elaine Harris (1-412-490-8065, Elaine.harris@fishersci.com ) for more assistance with our products.
  
  

Compare Our Quality. 

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

Marker Gene Accepts Major Credit Cards.

Place your orders now, using Master Card or Visa and save time and money!  Our Customer Assistance Staff can now accept either Master Card or Visa Credit Card orders, securely by telephone (toll-free) at 1-888-218-4062 (Domestic orders only).   We will continue to accept Institutional Purchase Orders for our products, online or by FAX at 1-541-342-1960.  International customers should contact us by e-mail, post or telephone for more information about International Distributors and ordering.  For information on pricing for individual products, or for a quote on bulk quantities of our products or kits, please contact our technical assistance staff at techservice@markergene.com.   We will be happy to assist you. 

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