Marker Gene Monthly Newsletter   

October, 2006

Volume 6, Number 10

© 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.

Measurement of T-Cell Activation using LacZ FACS.

The binding of the α / β T-cell antigen receptors (TCR) to small antigenic peptides that are bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) triggers a complex series of intracellular events that ultimately leads to T- cell functional activity and proliferation. T-cell activation requires clustering of a threshold number of T-cell receptors (TCRs) at the site of antigen presentation. Usually c alcium sensitive fluorescent dyes such as Fura-2 AM, Fluo-3 as well as [ 3 H]thymidine uptake or interleukin 2 (IL-2) secretion have been used as assays of T-cell activation. But these methods suffer from the drawback that they only measure the average activation state of the bulk population of cells. Though calcium-sensitive dyes can be used to examine individual cells, calcium increases are transitory, which makes it difficult to study the asynchronous activation of T cells by physiological ligands. Recently, several new methods have been developed, using marker gene constructs, to conveniently and reliably study ligand-induced activation of individual T cells.

The nuclear factor of activated T cell (NF-AT) element (-285 to -256 within the 5' IL-2 enhancer) has been iden tified as the key element in the transcriptional regulation of the IL-2 gene. In addition, an IL-2 promoter driven lacZ construct has also been used in a similar manner. In the human Jurkat T-cell line, the presence of a trimer of the NF-AT element has been shown to be sufficient to induce transcription of het erologous reporter genes in cells activated by phorbol ester and ionomycin . When this reporter construct is introduced into T-cell hybridomas, occupancy of the TCR triggers not only the secretion of IL-2 but also the production and intracellular accumulation of lacZ b -galactosidase which can be easily measured using fluorogenic substrates such as FDG ( Fluorescein di- b -D-Galactopyranoside , M0250 ).

The i ntracellular lacZ activity, a fter activation, is measured by loading the cells with FDG ( Fluorescein di- b -D-Galactoside , M0250 ) accord ing to the protocol described by Nolan, et al., (1988). Briefly, cells are centrifuged and washed twice in PBS containing 2% fetal bovine serum/10 mM HEPES, pH 7.4. Cells are then resuspended to a final volume of 0.1 ml in PBS and incubated for 5 min at 37°C, and loaded with FDG ( 0.1 ml of 1 mM FDG in water, prewarmed to 37°C). After a 1-min incubation at 37°C, 2 mL of ice-cold PBS/2% fetal bovine serum/ 10 mM Hepes is added and incubated on ice for 1 hr. before FACS analysis. For more information about these techniques, please see the references below, or visit our website.

• Karttunen, J., Shastri N., (1991) “Measurement of Ligand-Induced Activation in Single Viable T Cells Using the lacZ Reporter Gene” Proc. Natl. Acad. Sci. USA 88: 3972-3976.
• Kattunen, J., Sanderson, S., Shastri N., (1992) “Expression Cloning Strategy for T-Cell Antigens Detection of Rare Antigen-Presenting Cells by the lacZ T-Cell Activation Assay Suggests” Proc. Natl. Acad. Sci. USA 89: 6020-6024.
• Brombacher, F., Schäfer, T., Welssensteln, U., Tschopp, C., Andersen, E., Bürki K . , Bafmann G., (1994) “ IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice”, Intl. Immun. 6(2): 189-197.
• Shaw, J.-P., Utz, P. J., Durand, D. B., Toole, J. J., Emmel, E. A., Crabtree, G. R. (1988) Science 241: 202-205.
• Fiering, S., Northrop, J. P., Nolan, G. P., Matilla, P., Crab tree, G. R., Herzenberg, L.A. (1990) Genes Dev. 4: 1823- 1834.
• Nolan, G. P., Fiering, S., Nicolas, J.F. Herzenberg, L. A. (1988) Proc. Natl. Acad. Sci. USA 85: 2603-2607.
  

Biotin Labeling of Proteins, Nucleotides and Carbohydrates.

Direct labeling of proteins, oligonucleotides or carbohydrates with D-biotin is a very popular technique for analysis or quantification of these components in cellular, microplate, or in vitro high-throughput assay systems. Biotin labeling of antibodies eliminates the need to use a secondary antibody, providing lower background and higher sensitivity. The MarkerGene TM Biotin-X Antibody/Protein Labeling Kit (Product M1138) has been developed to allow for easy formation of desired biotin-protein, nucleotide or carbohydrate conjugates. The Biotin-X succinimidyl ester reagent ( M0783 ) provided in this kit forms the biotin-conjugates by reacting with non-protonated aliphatic amine groups, including the amine terminus of proteins and the e -amino group of lysines, nucleophilic groups on carbohydrates, and amino-nucleotide analogs. Biotin-X is routinely used for these labelings and has even been used for direct labeling of live cells, since it contains a long-chain linking arm that improves binding to avidin or streptavidin. The resulting biotin labeled proteins, peptides, glycolipids or carbohydrates can be visualized by reacting with fluorescently labeled avidin or streptavidin conjugates (like our Texas RedTM-Avidin , M1124 ) . This kit can be used with a wide range of protein concentrations and with nearly all proteins having a MW of 25 kD or higher. Biotin-X- antibody conjugates are useful in several applications including immunocytochemistry , flow cytometry, and fluorescence microscopy. The MarkerGene TM Biotin-X- Antibody/Protein Labeling Kit contains enough reagents for 5 protein labeling experiments or 25 cell labeling experiments; including buffers, solvents, gel-filtration chromatography media, and materials for a fast and easy biotin quantitation assay. The kit also includes a detailed, easy to follow protocol. For more information, please visit our website , or take a look at the Biotin-X Antibody/Protein Labeling Kit Product Page .

• Perrier A.L., Massoulie J., Krejci E., (2002) "PRiMA: the membrane anchor of acetylcholinesterase in the brain." Neuron. 33: 275-285.
• Soukup G.A., Cerny R.L., Maher L.J., (1995) "Preparation of oligonucleotide-biotin conjugates with cleavable linkers." Bioconjug Chem 6: 135-138.
• Abel A.P., Weller M.G., Duveneck G.L., Ehrat M., Widmer H.M., (1996) "Fiber-optic evanescent wave biosensor for the detection of oligonucleotides." Anal Chem 68: 2905-2912.
• Peng H.B., Xie H, Rossi S.G., Rotundo R.L., (1999) "Acetylcholinesterase clustering at the neuromuscular junction involves perlecan and dystroglycan." J Cell Biol. 145: 911-921.
• Haugland, R. P., Bhalgat, M. K. (1998) “Preparation of avidin conjugates.” Methods Mol. Biol. 80: 185-196.
• Haugland, R.P., You, W. W. (1998) “Coupling of antibodies with biotin.” Methods Mol. Biol. 80: 173-183.
• Haugland R.P., You W.W., (1995) “Coupling of monoclonal antibodies with biotin.” Methods Mol Biol. 45: 223-233.
• Green, N.M. (1970) “Spectrophotometric Determination of Avidin and Biotin.” Meth. Enzymol. 18: 418-425.

Non-Ionic Detergents for Membrane Biochemistry.

A variety of non-ionic detergents are available for purification of membrane proteins, lysis of cells or stabilization, crystallization, or even denaturation of lipophilic proteins or membrane factors. Non-ionic detergent can also be used for purification of soluble recombinant proteins from cells, including bacteria, and for solubilization and reconstitution of lipophilic proteins. In addition, these detergents have been used to aide in the extraction of DNA or RNA, the preparation of liposomes, the prevention of precipitation from solutions, increasing the resolution of proteins in 2D gels and the prevention of non-specific binding in immunoassays. Among the most popular detergents are Triton X-100 (Polyethylene glycol (10), tert-octylphenyl ether), Tween-20 (Polyoxyethylene (5) sorbitan monolaurate) and Octyl b - D -glucoside (octyl glucoside). Tween-20 and Triton X-100 can be somewhat harsh and denaturing on membrane or soluble proteins (similar to SDS), whereas octyl glucoside is often used as a milder reagent that has become more accepted for these purposes. Octyl glucoside itself, however, is subject to enzymatic degradation in biological or cellular samples, and a new, non-ionic, non-denaturing detergent for the solubilization and reconstitution of membrane proteins, octyl 1-thio- b -D-glucopyranside ( octyl thioglucoside , M1228 ) has been found to be much more stable than the frequently used octyl- ß -glucoside for these applications. In addition, its low molecular weight allows for easy removal by dialysis. For more information about these detergents, lysis buffers, and their uses, please visit our website or see the references below.

• Saito, S., Tsuchiya, T., (1984) "Characteristics of n-octyl ß -D-thioglucopyranoside, a new non-ionic detergent useful for membrane biochemistry." Biochem. J. 222: 829.
• Yoshida, M., (1994) "Dissociation of the complex of dystrophin and its associated proteins into several unique groups by n-octyl ß -D-glucoside." Eur. J. Biochem. 222: 1055-1061.
• Stubbs G.W., (1976) “Nonionic detergent with no absorbance at 228 µ m, best suited for the solubilisation and isolation of membrane proteins.” Biochim. Biophys. Acta 426: 46.
• Holloway, P.J., Arundel, P.H., (1988) “High-resolution two-dimensional electrophoresis of plant proteins.” Anal. Biochem. 172: 8-15.
• Gould, R.J., Ginsberg B.H., Spector A.A.,  (1981). “Effects of octyl beta-glucoside on insulin binding to solubilized membrane receptors.” Biochemistry 20: 6776-6781.
• Jackson , M.L., Schmidt C.F., Lichtenberg D., Litman B.J., Albert A.D.,  (1982). “ Solubilization of phosphatidylcholine bilayers by octyl glucoside.” Biochemistry 21 : 4576-4582.
• Rosevear, P., VanAken T., Baxter J., Ferguson-Miller S., (1980). “Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase.” Biochemistry 19: 4108-4115.
• Jackson M.L., Litman B.J., (1982) “Rhodopsin-phospholipid reconstitution by dialysis removal of octyl glucoside” Biochemistry . 21(22): 5601-8.

High-Throughput Microarray Lipase Assay Systems.

Recent work from the laboratory of Professor Jean-Louis Reymond at the Department of Chemistry and Biochemistry, University of Berne – Switzerland has identified several new systems for high-throughput assay of multiple enzyme activities by adsorbing various substrates onto amine-functionalized glass slides coated with bovine serum albumin and a short PEG linker. This substrate is then reacted with the molecule (5R)/(5S)-3-(5,6-dihydroxyhexyloxy)benzaldehyde by reductive alkylation. Onto the hydroxyl groups can be esterifed various aliphatic groups specific for multiple lipase activities (C-2 through C-12 fatty acids, for example). These 1,2-diol mono or di-esters allow classification of a variety of lipases or esterases according to the acyl chain length dependence of their reactivity. Once the lipase activity has occurred, the resulting diol is detected by NaIO 4 -oxidation of the 1,2-diol hydrolysis product, which forms an aldehyde. The aldehyde can then be tagged using an aldehyde reactive fluorescent dye, like the red-fluorescent dye rhodamine B sulfohydrazide, fluorescein-5-semithiocarbazide ( M01036 ) or NBD-hydrazide ( M1025 ). Specific fingerprints are produced by active enzymes. These experiments provide the first example of lipase fingerprinting using microarray analyses.

In a similar manner, attaching the 3,4-dihydroxypropylether function to various fluorophores, followed by esterification with specific acyl groups, produces similar specific esterase or lipase substrates that produce their corresponding dyes upon enzyme activity and periodate oxidation (immodulating linking group). For more information about these new assays, please see the references below or contact us at techservice@markergene.com .

• Grognux, J., Reymond, J.L., (2006) “A red-fluorescent substrate microarray for lipase fingerprinting.” Mol. Biosys . 2: 492-498.
• Yang, Y., Babiak, P., Reymond, J.L., (2006) “New Monofunctionalized Fluorescein Derivatives for the Efficient High-Throughput Screening of Lipases and Esterases in Aqueous Media.” Helv. Chim. Acta 89: 404-415.
• Yang, Y., Babiak, P., Reymond, J.L., (2006) “Low background FRET- substrates for lipases and esterases suitable for high-throughput screening under basic (pH 11) conditions.” Org. Biomol. Chem . 4: 1746-1754.
• http://dcbwww.unibe.ch/groups/reymond/

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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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