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Marker Gene Monthly Newsletter

June, 2005

Volume 5, Number 6

© 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.

Peptidase Assays Using Aminoluciferin Light-Up.

Several new bioluminescent protease assays have been developed that employ peptides or amino acids conjugated to 6-amino-D-luciferin (M0352) for analysis of a variety of peptidase enzymes including various caspases and cathepsins, a-chymotrypsin, trypsin, elastase, kallikrein, thrombin, ficin, bromoalain, plasmin, papain, ficin, and many others or for use in vivo tracing of luciferase transfected cells in living tissues. These assays produce the 6-amino analog of luciferin which displays a bioluminescence spectrum similar to that observed for the natural substrate D-luciferin. The background often found in fluorescence based peptidase assays is minimized, and detection limits are up to 2 orders of magnitude more sensitive. Homogeneous assays require two enzymes; peptidase-catalyzed hydrolysis of the substrate to release 6-amino-D-luciferin, which in turn serves as the substrate for the second enzyme, firefly luciferase. 6-Amino-D-luciferin is not the natural substrate for firefly luciferase, but is accepted by the enzyme as a substrate, and enzyme analysis with luciferase uses the same protocol. In addition, these peptidase assays using a bioluminogenic substrate can be easily adapted to 96-well formats for HTS-based assays. The general principle of coupling peptidase activity to luciferase activity using a peptide amino-D-luciferin conjugate was first demonstrated by Monsees, T., et al. Using this principle, it should be possible to detect any peptidase or protease activity via bioluminescence by using a substrate with a suitable peptide sequence. Applications in pathogen detection, discovery of protease inhibitors, probing cell physiology and assessing protease activity in oncogenesis are possible at extraordinary sensitivity. Marker Gene now manufactures two extremely useful starting materials for peptidase substrate synthesis applications; the substrate (M0352) 6-amino-D-luciferin as well as the protected version 6-N-FMOC-D-Luciferin (M0802) for solid-phase peptide synthesis applications, and can help you develop your own ultrasensitive chemiluminescent peptidase assays. For more information about these assays, see the references below or visit our website.

M. A. O’Brien, P. E. Hesselberth, W. J. Daily, M. A. Scurria, D. H. Klaubert, R. F. Bulleit, K. V. Wood, (2005) “Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model” Journal of Biomolecular Screening 10(2): 137-148.

R. Shinde, C.H. Contag, J. Perkins, “In vivo Protease Assay Imaging Using Aminoluciferyl Derivatives” http://biophotonics.ucdavis.edu/events/retreat04/Shindes.ppt

K. Shah, C.-H. Tung, X. O. Breakefield, R. Weissleder “In Vivo Imaging of S-TRAIL-Mediated Tumor Regression and Apoptosis” MOLECULAR THERAPY 11( 6) 926-931.

Mishra, R., Emancipator, S.N., Kern, T., Simonson, M.S., (2005) “High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells” Kidney Intl. 67(1): 82.

Monsees T., Geiger R., Miska W., (1995) “A novel bioluminogenic assay for alpha-chymotrypsin.” J Biolumin Chemilumin. 10(4): 213-8

White, E. H.; Woerther, H., Seliger, H. H., McElroy, W. D. (1966) “Amino analogs of firefly luciferin and biological activity thereof.” Journal of the American Chemical Society 88(9): 2015-18.

Denburg, Jeffrey; Lee, Reiko Takasaka; McElroy, W. D. (1969) “Substrate-binding properties of firefly luciferase. I. Luciferin-binding site. “ Archives of Biochemistry and Biophysics 134(2): 381-94.

Geiger, R., Miska, W., “Aminoluciferin derivatives, processes for the production thereof and their application in the determination of enzyme activities.” US Patent 5035999 (1991).

Monsees, Thomas; Miska, Werner; Geiger, Reinhard. (1994) “Synthesis and characterization of a bioluminogenic substrate for a-chymotrypsin.” Anal. Biochem. 221(2): 329-34.

Flow cytometry (FACS) lacZ b-Galactosidase Assay.
Marker Gene is developing selection strategies that impart a growth advantage specifically to cells that express lacZ b-galactosidase. However, cloned lacZ b-galactosidase can be detected in live cells using fluorescent substrates, and fluorescent cells can be efficiently separated from mixed populations rapidly and efficiently using flow cytometry without affecting their viability. This assay is relatively simple and can be performed in less than an hour when the number of samples to be analyzed is limited. Furthermore, it is quantitative and can be routinely used to follow the kinetics of a given interaction in response to specific signals. The traditional substrate used for this assay, fluorescein di-b-D-galactopyranoside (FDG, M0250) is not very cell permeable but can be introduced into the cells by hypotonic shock. Cleavage by beta-galactosidase results in the production of free fluorescein, which is generally unable to cross the plasma membrane and becomes trapped inside the beta-gal positive cells. To perform the assay, the cells to be analyzed are trypsinized, resuspended in phosphate buffered saline (PBS) containing 5% fetal bovine serum (PBS/FBS) and pelleted in a polystyrene round-bottom tube. The cells are then resuspended in 100 mL of PBS/FBS and an equal volume of bi-distilled water containing the substrate at a final concentration of 1 mM is added. After three minutes at room temperature, the hypotonic conditions are quenched by adding ten volumes of ice-cold PBS/FBS containing 1mg/ml Propidium Iodine (PI). PI is a red fluorescent compound that is actively excluded from living cells but accumulates in dead cells, allowing their exclusion from the analysis or the sorting. The cells are pelleted again, resuspended in approximately 200 ml of ice cold PBS/FBS and analyzed on a Becton-Dickinson FACScan or sorted on a Becton-Dickinson FACStar flow cytometer. A powerful advantage of this FACS assay is its ability to monitor protein interactions in live cells using a Two-Hybrid system, as it provides a simple means of selecting for cells in which a given interaction takes place. This opens the door for screening cDNA libraries for novel interaction partners for a given "target" protein in mammalian cells. The FACS method of selection will be most likely superior to growth-based selection protocols, as many interactions are likely to be transient under physiological conditions. Marker Gene sells all the reagents and also several kits for FACS-Gal analysis including the In vivo lacZ b-Galactosidase Intracellular Detection Kit (M0259) and the FACS Blue lacZ b-Galactosidase Detection Kit (M0255). Please see the references below and visit our website for more information.
K. Abe, M. Hashiyama, G. Macgregor, K. Yamamura, K. Abe (1996) “Purification of Primordial Germ Cells from TNAP^-geoMouse Embryos Using FACS-gal” Devel. Biol. 80(2): 468-472.

F. Rossi, C.A. Charlton, H. M. Blau (1997) “Monitoring protein-protein interactions in intact eukaryotic cells by b-galactosidase complementation” Proc. Natl. Acad. Sci. USA 94: 8405-8410.

F. Brombacher, T. Schafer, U. Weissenstein, C. Tschopp, E. Andersen, K. Burki and G. Baumann (1994) “IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice” Intl. Imunol. 6: 189-197.

Saalmuller A, Mettenleiter TC. (1993) "Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based beta-galactosidase assay and flow cytometry." J Virol. Methods 44: 99.

Roederer M, Fiering S, Herzenberg LA. (1991) "FACS-Gal: flow cytometric analysis and sorting of cells expressing reporter gene constructs." Methods: Comp. Meth. Enzymol. 2: 248.

Kerr WG, Herzenberg LA. (1991) "Gene-search viruses and FACS-Gal permit the detection, isolation, and characterization of mammalian cells with in situ fusions between cellular genes and escherichia coli lacZ." Methods: Comp. Meth. Enzymol. 2: 261.

New Protein Mobility Probe.

The new eosin analog, eosin-5-isothiocyanate (EITC) (M1074), is an effective phosphorescent probe for monitoring the rotational properties of single proteins, protein assemblies, membrane proteins, and other biomolecules using phosphorescence anisotropy. Eosin is a brominated analog of fluorescein that yields 20 times more oxygen than fluorescein upon excitation and is also an effective photooxidizer of Diaminobenzidine (DAB) in high resolution electron microscopy studies. Furthermore, the small size of eosin allows for exceptional tissue penetration, resulting in increased electron microscopy resolution. Eosin derivatives, such as EITC can also be used as acceptors in fluorescence resonance energy transfer (FRET) studies (eosin EX/EM: 520/544nm) due to efficient fluorescence absorption overlap with fluorescein. Marker Gene is currently developing a new EITC Protein Mobility Labeling Kit (M1081), which provides reagents, standards and a detailed protocol for up to five protein labeling experiments using simple spin column purification. For more information, see the references below or visit our website.

"General Strategy for the Synthesis of Eosin Fluorescein Energy Transfer Substrates for High Sensitivity Screening of Protease Inhibitors." Contillo LG, et al. Techniques in Protein Chemistry V, Crabb JW, Ed. pp. 493-500 (1994).

"Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy." Hulspas R, Krijtenburg PJ, Keij JF, Bauman JG. J Histochem Cytochem 41: 1267-1272 (1993).

"The Binding of Eosin-Labeled Subunit S to the Isolated Chloroplast ATPase, CF1, as Revealed by Rotational Diffusion in Solution." Wagner R, et al. FEBS Lett 230: 109 (1988).

"Chemical Modification of the Phosphoenolpyruvate Carboxylase from Maize Leaves and Its Conformation in Isotropic Solution. Studies Via Triplet Lifetime and Rotational Diffusion Using Eosin Isothiocyanate as Label." Andreo CS, et al. Biochim Biophys Acta 870: 292 (1986).

"Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte." Chiba T, Sato Y, Suzuki Y. Biochim Biophys Acta 858: 107-117 (1986).

"Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins." Johnson P, Garland PB. Biochem J 203: 313-321 (1982).

"Coupling factor for photophosphorylation labeled with eosin isothiocyanate: activity, size, and shape in solution." Wagner R, Junge W. Biochemistry 21: 1890-1899 (1982).

"Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase." Nigg E, Kessler M, Cherry RJ. Biochim Biophys Acta 550: 328-340 (1979).

New Cholinesterase Detection Kit.

Cholinesterase enzyme activities (acetylcholinesterase and butyrylcholinesterase) are widely used for the study and treatment of cholinergic or neurotransmission-related diseases such as Alzheimer’s Disease (AD). As AD progresses, acetylcholinesterase (AChE) activity decreases in some brain regions, while butyrylcholinesterase (BChE) activity increases. Both AChE and BChE are associated with amyloid and neurofibrillary tangles in the brains of elderly persons with, and without, relevant cognitive impairment. It is important to identify what factors may contribute to the transformation of “benign amyloid” to one capable of causing the classical neurological symptoms of the AD state. BChE is a possible factor in this transformation process. Advanced amyloid plaques in Alzheimer brains have up to 87% BChE reactivity, compared with 20% reactivity in early, benign deposits. The presence of BChE activity may distinguish the neuro-toxic plaques from those seen in normal aging since BChE activity appears to play a role in the transformation of benign amyloid plaques into the forms associated with neuronal degeneration and clinical dementia. In addition, AChE activity has previously been detected in several different apoptotic cell lines and is proposed to be associated with the apoptosome formation during the initial stage of apoptosis. Marker Gene has teamed up with Immunochemistry Technologies, Inc. (Bloomington, MN) to develop a new assay system for distinquishing acetylcholinesterase and butrylcholinesterase activities in live cells. These assays are based upon a new physostigmine-based inhibitor that is specific for the active site of these enzymes.

In this new assay, fluorescein has been conjugated to a physostigmine analog, eseroline, through a 5-carbon spacer linked via a carbamoyl carbonyl group on the physostigmine side chain. The resulting conjugate (Physostigmine – Fluorescein, Ph- Fl) is highly specific and can be used to detect the activity of these important cholinesterase enzymes, as well as apoptosis induction in several different cell lines. For more information about these new inhibitors, cholinesterase and apoptosis assays please visit our website or see the references below.

Park, S. E., Kim, N. D., Yoo Y. H., (2004). “Acetylcholinesterase plays a pivotal role in apoptosome formation.” Cancer Res. 64: 2652-2655.

Zhang, X. J., Yang, L. Zhao Q., Caen, J. P. He, H. Y. Jin, Q. H. Guo, L. H. Alemany, M. Zhang, L. Y. Shi Y. F. (2002) “Induction of acetylcholinesterase expression during apoptosis in various cell types.” Cell Death and Differ. 9: 790- 800.

Atack, J. R., Yu, QS, Soncrant, T.T., Brossi, A., Rapoport, S. I. (1989.) “Comparative inhibitory effects of various physostigmine analogs against acetyl- and butyrylcholinesterases.” J. Pharmacol. Exp. Ther. 249(1): 194-202.

Herholz, K., Bauer, B. Wienhard, K. Kracht, L. Mielke, R. Lenz, M. O. Strotmann, T.., Heiss, W. D., (2000) “In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.” J. Neural. Transm. 107(12): 1457-1468.

Giacobini, E. (2004) “Cholinesterase inhibitors: new roles and therapeutic alternatives.” Pharmacol. Res. 50(4): 433-440.

Giacobini, E. (2003) “Cholinergic function and Alzheimer’s disease.” Int. J. Geriatr. Psychiatry 18(1): S1-5.

Ballard, C. G. (2002) “Advances in the treatment of Alzheimer’s disease: benefits of dual cholinesterase inhibition.” Eur. Neurol. 47: 64-70.

Yu, Q S., Zhu, X., Holloway, H. W., Whittaker, N. F., Brossi, A., Greig. N. H. (2002) “Anticholinesterase Activity of Compounds related to geneserine tautomers, N-oxides and 1,2-oxazines.” J. Med. Chem. 45: 3684-3691.

Huang, X., Lee, B., Johnson, G., Naleway, J., Guzikowski, A., Dai, W., Darzynkiewicz, Z., (2005) “Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to In Situ detect active acetylcholinesterase (AChE) induced during apoptosis. Cell Cycle 4(1): 140-147.

Fluorescent Lysyl Oxidase Assay Systems.

Lysyl oxidases (EC 1.4.3.13, protein-lysine 6-oxidases, LOX) are extracellular copper enzymes that initiate the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues. The cross-links formed are responsible for the tensile strength of collagen fibers, the unique elastic properties of elastin and overall ECM maturation. Four human lysyl oxidase (LOX) isoenzymes have been identified and characterized. The LOXL3 gene is expressed in several tissues, the highest expression levels being in the placenta, heart, ovary, testis, small intestine, and spleen. The LOXL4 gene is likewise expressed in most human tissues studied, the highest levels being seen in the skeletal muscle, testis, and pancreas. LOX proteins have been shown to be secreted extracellular proteins. LOX is critically required for the biosynthesis of functional extracellular matrices. However, excessive accumulation of insoluble collagen fibers also leads to fibrosis, and up-regulation of LOX expression and increased LOX activity is found in several fibrotic diseases. Development of specific inhibitors of LOX activity with the ultimate goal of preventing excessive formation of insoluble collagenous extracellular matrices is a potential therapeutic strategy to treat these fibrotic diseases. Lysyl oxidase is also known for its tumor suppressor activity and decreased levels of lysyl oxidase mRNA are found in most cancer cell lines as well as in murine cells transformed by several oncogenes. Few specific assay systems are available for LOX. Originally, a tritium-based assay was used to measure LOX activity. It involved the purification of LOX and tritium radio-labeled lysine or tropoelastin that then served as a substrate for LOX. This method does not work well to measure LOX activity in cell lysates or in animal tissues and homogenates. Recently several LOX assay systems have been developed utilizing the peroxide by-product of reaction. The reaction for lysyl oxidase proceeds as follows as it reacts with primary amines: RCH₂NH₂ + O₂ ^--à RCHO^- + NH₃ +_.Horseradish peroxidase with homovanillate and Amplex Red have both been used to try to measure the activity of LOX utilizing the H₂O₂ produced, but neither has proved to be reliable and reproducible. Marker Gene now provides several sensitive substrates that should be useful for fluorescence detection of LOX activity, including dihydrorhodamine 123 (M0545) (peroxide sensitive), biocytin hydrazide (M0128), fluorescein-5-semithiocarbazide (M01036) and NBD-hydrazide (M1025) (aldehyde labeling). For more information about these assays and techniques, please visit our website or check the references below.

Palamakumbura AH, Trackman PC. (2002) “A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples.” Anal Biochem. 300(2): 245-51.

X. Liu, Y. Zhao, J. Gao, B. Pawlyk, B. Starcher, J. A. Spencer, H. Yanagisawa, J. Zuo, T. Li, (2004) “Elastic fiber homeostasis requires lysyl oxidase−like 1 protein” Nature Genetics 36: 178-182.

Wakasaki H., Ooshima A., (1990) “Immunohistochemical localization of lysyl oxidase with monoclonal antibodies.” Lab Invest. 63(3): 377-384.

Kagan, H. M., Sullivan, K. A. (1982) “Lysyl oxidase: Preparation and role in elastin biosynthesis.” Methods Enzymol. 82: 637–650.

Trackman, P. C., Zoski, C. G., Kagan, H. M. (1981) Development of a peroxidase-coupled fluorometric assay for lysyl oxidase. Anal. Biochem. 113: 336–342.

Zhou, M., Diwu, Z., Panchuk-Voloshina, N., Haugland, R. P. (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases.” Anal. Biochem. 253: 162–168.

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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	If you are having trouble viewing this email, click on this link. To order any of our products, please go to our ORDER FORM or visit one of our distributors:
	Marker Gene Monthly Newsletter June, 2005 Volume 5, Number 6 © 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.
	Peptidase Assays Using Aminoluciferin Light-Up. Several new bioluminescent protease assays have been developed that employ peptides or amino acids conjugated to 6-amino-D-luciferin (M0352) for analysis of a variety of peptidase enzymes including various caspases and cathepsins, a-chymotrypsin, trypsin, elastase, kallikrein, thrombin, ficin, bromoalain, plasmin, papain, ficin, and many others or for use in vivo tracing of luciferase transfected cells in living tissues. These assays produce the 6-amino analog of luciferin which displays a bioluminescence spectrum similar to that observed for the natural substrate D-luciferin. The background often found in fluorescence based peptidase assays is minimized, and detection limits are up to 2 orders of magnitude more sensitive. Homogeneous assays require two enzymes; peptidase-catalyzed hydrolysis of the substrate to release 6-amino-D-luciferin, which in turn serves as the substrate for the second enzyme, firefly luciferase. 6-Amino-D-luciferin is not the natural substrate for firefly luciferase, but is accepted by the enzyme as a substrate, and enzyme analysis with luciferase uses the same protocol. In addition, these peptidase assays using a bioluminogenic substrate can be easily adapted to 96-well formats for HTS-based assays. The general principle of coupling peptidase activity to luciferase activity using a peptide amino-D-luciferin conjugate was first demonstrated by Monsees, T., et al. Using this principle, it should be possible to detect any peptidase or protease activity via bioluminescence by using a substrate with a suitable peptide sequence. Applications in pathogen detection, discovery of protease inhibitors, probing cell physiology and assessing protease activity in oncogenesis are possible at extraordinary sensitivity. Marker Gene now manufactures two extremely useful starting materials for peptidase substrate synthesis applications; the substrate (M0352) 6-amino-D-luciferin as well as the protected version 6-N-FMOC-D-Luciferin (M0802) for solid-phase peptide synthesis applications, and can help you develop your own ultrasensitive chemiluminescent peptidase assays. For more information about these assays, see the references below or visit our website. M. A. O’Brien, P. E. Hesselberth, W. J. Daily, M. A. Scurria, D. H. Klaubert, R. F. Bulleit, K. V. Wood, (2005) “Homogeneous, Bioluminescent Protease Assays: Caspase-3 as a Model” Journal of Biomolecular Screening 10(2): 137-148. R. Shinde, C.H. Contag, J. Perkins, “In vivo Protease Assay Imaging Using Aminoluciferyl Derivatives” http://biophotonics.ucdavis.edu/events/retreat04/Shindes.ppt K. Shah, C.-H. Tung, X. O. Breakefield, R. Weissleder “In Vivo Imaging of S-TRAIL-Mediated Tumor Regression and Apoptosis” MOLECULAR THERAPY 11( 6) 926-931. Mishra, R., Emancipator, S.N., Kern, T., Simonson, M.S., (2005) “High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells” Kidney Intl. 67(1): 82. Monsees T., Geiger R., Miska W., (1995) “A novel bioluminogenic assay for alpha-chymotrypsin.” J Biolumin Chemilumin. 10(4): 213-8 White, E. H.; Woerther, H., Seliger, H. H., McElroy, W. D. (1966) “Amino analogs of firefly luciferin and biological activity thereof.” Journal of the American Chemical Society 88(9): 2015-18. Denburg, Jeffrey; Lee, Reiko Takasaka; McElroy, W. D. (1969) “Substrate-binding properties of firefly luciferase. I. Luciferin-binding site. “ Archives of Biochemistry and Biophysics 134(2): 381-94. Geiger, R., Miska, W., “Aminoluciferin derivatives, processes for the production thereof and their application in the determination of enzyme activities.” US Patent 5035999 (1991). Monsees, Thomas; Miska, Werner; Geiger, Reinhard. (1994) “Synthesis and characterization of a bioluminogenic substrate for a-chymotrypsin.” Anal. Biochem. 221(2): 329-34.
	Flow cytometry (FACS) lacZ b-Galactosidase Assay. Marker Gene is developing selection strategies that impart a growth advantage specifically to cells that express lacZ b-galactosidase. However, cloned lacZ b-galactosidase can be detected in live cells using fluorescent substrates, and fluorescent cells can be efficiently separated from mixed populations rapidly and efficiently using flow cytometry without affecting their viability. This assay is relatively simple and can be performed in less than an hour when the number of samples to be analyzed is limited. Furthermore, it is quantitative and can be routinely used to follow the kinetics of a given interaction in response to specific signals. The traditional substrate used for this assay, fluorescein di-b-D-galactopyranoside (FDG, M0250) is not very cell permeable but can be introduced into the cells by hypotonic shock. Cleavage by beta-galactosidase results in the production of free fluorescein, which is generally unable to cross the plasma membrane and becomes trapped inside the beta-gal positive cells. To perform the assay, the cells to be analyzed are trypsinized, resuspended in phosphate buffered saline (PBS) containing 5% fetal bovine serum (PBS/FBS) and pelleted in a polystyrene round-bottom tube. The cells are then resuspended in 100 mL of PBS/FBS and an equal volume of bi-distilled water containing the substrate at a final concentration of 1 mM is added. After three minutes at room temperature, the hypotonic conditions are quenched by adding ten volumes of ice-cold PBS/FBS containing 1mg/ml Propidium Iodine (PI). PI is a red fluorescent compound that is actively excluded from living cells but accumulates in dead cells, allowing their exclusion from the analysis or the sorting. The cells are pelleted again, resuspended in approximately 200 ml of ice cold PBS/FBS and analyzed on a Becton-Dickinson FACScan or sorted on a Becton-Dickinson FACStar flow cytometer. A powerful advantage of this FACS assay is its ability to monitor protein interactions in live cells using a Two-Hybrid system, as it provides a simple means of selecting for cells in which a given interaction takes place. This opens the door for screening cDNA libraries for novel interaction partners for a given "target" protein in mammalian cells. The FACS method of selection will be most likely superior to growth-based selection protocols, as many interactions are likely to be transient under physiological conditions. Marker Gene sells all the reagents and also several kits for FACS-Gal analysis including the In vivo lacZ b-Galactosidase Intracellular Detection Kit (M0259) and the FACS Blue lacZ b-Galactosidase Detection Kit (M0255). Please see the references below and visit our website for more information. K. Abe, M. Hashiyama, G. Macgregor, K. Yamamura, K. Abe (1996) “Purification of Primordial Germ Cells from TNAP^-geoMouse Embryos Using FACS-gal” Devel. Biol. 80(2): 468-472. F. Rossi, C.A. Charlton, H. M. Blau (1997) “Monitoring protein-protein interactions in intact eukaryotic cells by b-galactosidase complementation” Proc. Natl. Acad. Sci. USA 94: 8405-8410. F. Brombacher, T. Schafer, U. Weissenstein, C. Tschopp, E. Andersen, K. Burki and G. Baumann (1994) “IL-2 promoter-driven lacZ expression as a monitoring tool for IL-2 expression in primary T cells of transgenic mice” Intl. Imunol. 6: 189-197. Saalmuller A, Mettenleiter TC. (1993) "Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based beta-galactosidase assay and flow cytometry." J Virol. Methods 44: 99. Roederer M, Fiering S, Herzenberg LA. (1991) "FACS-Gal: flow cytometric analysis and sorting of cells expressing reporter gene constructs." Methods: Comp. Meth. Enzymol. 2: 248. Kerr WG, Herzenberg LA. (1991) "Gene-search viruses and FACS-Gal permit the detection, isolation, and characterization of mammalian cells with in situ fusions between cellular genes and escherichia coli lacZ." Methods: Comp. Meth. Enzymol. 2: 261.
	New Protein Mobility Probe. The new eosin analog, eosin-5-isothiocyanate (EITC) (M1074), is an effective phosphorescent probe for monitoring the rotational properties of single proteins, protein assemblies, membrane proteins, and other biomolecules using phosphorescence anisotropy. Eosin is a brominated analog of fluorescein that yields 20 times more oxygen than fluorescein upon excitation and is also an effective photooxidizer of Diaminobenzidine (DAB) in high resolution electron microscopy studies. Furthermore, the small size of eosin allows for exceptional tissue penetration, resulting in increased electron microscopy resolution. Eosin derivatives, such as EITC can also be used as acceptors in fluorescence resonance energy transfer (FRET) studies (eosin EX/EM: 520/544nm) due to efficient fluorescence absorption overlap with fluorescein. Marker Gene is currently developing a new EITC Protein Mobility Labeling Kit (M1081), which provides reagents, standards and a detailed protocol for up to five protein labeling experiments using simple spin column purification. For more information, see the references below or visit our website. "General Strategy for the Synthesis of Eosin Fluorescein Energy Transfer Substrates for High Sensitivity Screening of Protease Inhibitors." Contillo LG, et al. Techniques in Protein Chemistry V, Crabb JW, Ed. pp. 493-500 (1994). "Avidin-EITC: an alternative to avidin-FITC in confocal scanning laser microscopy." Hulspas R, Krijtenburg PJ, Keij JF, Bauman JG. J Histochem Cytochem 41: 1267-1272 (1993). "The Binding of Eosin-Labeled Subunit S to the Isolated Chloroplast ATPase, CF1, as Revealed by Rotational Diffusion in Solution." Wagner R, et al. FEBS Lett 230: 109 (1988). "Chemical Modification of the Phosphoenolpyruvate Carboxylase from Maize Leaves and Its Conformation in Isotropic Solution. Studies Via Triplet Lifetime and Rotational Diffusion Using Eosin Isothiocyanate as Label." Andreo CS, et al. Biochim Biophys Acta 870: 292 (1986). "Amino acid residues complexed with eosin 5-isothiocyanate in band 3 protein of the human erythrocyte." Chiba T, Sato Y, Suzuki Y. Biochim Biophys Acta 858: 107-117 (1986). "Fluorescent triplet probes for measuring the rotational diffusion of membrane proteins." Johnson P, Garland PB. Biochem J 203: 313-321 (1982). "Coupling factor for photophosphorylation labeled with eosin isothiocyanate: activity, size, and shape in solution." Wagner R, Junge W. Biochemistry 21: 1890-1899 (1982). "Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements: inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase." Nigg E, Kessler M, Cherry RJ. Biochim Biophys Acta 550: 328-340 (1979).
	New Cholinesterase Detection Kit. Cholinesterase enzyme activities (acetylcholinesterase and butyrylcholinesterase) are widely used for the study and treatment of cholinergic or neurotransmission-related diseases such as Alzheimer’s Disease (AD). As AD progresses, acetylcholinesterase (AChE) activity decreases in some brain regions, while butyrylcholinesterase (BChE) activity increases. Both AChE and BChE are associated with amyloid and neurofibrillary tangles in the brains of elderly persons with, and without, relevant cognitive impairment. It is important to identify what factors may contribute to the transformation of “benign amyloid” to one capable of causing the classical neurological symptoms of the AD state. BChE is a possible factor in this transformation process. Advanced amyloid plaques in Alzheimer brains have up to 87% BChE reactivity, compared with 20% reactivity in early, benign deposits. The presence of BChE activity may distinguish the neuro-toxic plaques from those seen in normal aging since BChE activity appears to play a role in the transformation of benign amyloid plaques into the forms associated with neuronal degeneration and clinical dementia. In addition, AChE activity has previously been detected in several different apoptotic cell lines and is proposed to be associated with the apoptosome formation during the initial stage of apoptosis. Marker Gene has teamed up with Immunochemistry Technologies, Inc. (Bloomington, MN) to develop a new assay system for distinquishing acetylcholinesterase and butrylcholinesterase activities in live cells. These assays are based upon a new physostigmine-based inhibitor that is specific for the active site of these enzymes. In this new assay, fluorescein has been conjugated to a physostigmine analog, eseroline, through a 5-carbon spacer linked via a carbamoyl carbonyl group on the physostigmine side chain. The resulting conjugate (Physostigmine – Fluorescein, Ph- Fl) is highly specific and can be used to detect the activity of these important cholinesterase enzymes, as well as apoptosis induction in several different cell lines. For more information about these new inhibitors, cholinesterase and apoptosis assays please visit our website or see the references below. Park, S. E., Kim, N. D., Yoo Y. H., (2004). “Acetylcholinesterase plays a pivotal role in apoptosome formation.” Cancer Res. 64: 2652-2655. Zhang, X. J., Yang, L. Zhao Q., Caen, J. P. He, H. Y. Jin, Q. H. Guo, L. H. Alemany, M. Zhang, L. Y. Shi Y. F. (2002) “Induction of acetylcholinesterase expression during apoptosis in various cell types.” Cell Death and Differ. 9: 790- 800. Atack, J. R., Yu, QS, Soncrant, T.T., Brossi, A., Rapoport, S. I. (1989.) “Comparative inhibitory effects of various physostigmine analogs against acetyl- and butyrylcholinesterases.” J. Pharmacol. Exp. Ther. 249(1): 194-202. Herholz, K., Bauer, B. Wienhard, K. Kracht, L. Mielke, R. Lenz, M. O. Strotmann, T.., Heiss, W. D., (2000) “In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.” J. Neural. Transm. 107(12): 1457-1468. Giacobini, E. (2004) “Cholinesterase inhibitors: new roles and therapeutic alternatives.” Pharmacol. Res. 50(4): 433-440. Giacobini, E. (2003) “Cholinergic function and Alzheimer’s disease.” Int. J. Geriatr. Psychiatry 18(1): S1-5. Ballard, C. G. (2002) “Advances in the treatment of Alzheimer’s disease: benefits of dual cholinesterase inhibition.” Eur. Neurol. 47: 64-70. Yu, Q S., Zhu, X., Holloway, H. W., Whittaker, N. F., Brossi, A., Greig. N. H. (2002) “Anticholinesterase Activity of Compounds related to geneserine tautomers, N-oxides and 1,2-oxazines.” J. Med. Chem. 45: 3684-3691. Huang, X., Lee, B., Johnson, G., Naleway, J., Guzikowski, A., Dai, W., Darzynkiewicz, Z., (2005) “Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to In Situ detect active acetylcholinesterase (AChE) induced during apoptosis. Cell Cycle 4(1): 140-147.
	Fluorescent Lysyl Oxidase Assay Systems. Lysyl oxidases (EC 1.4.3.13, protein-lysine 6-oxidases, LOX) are extracellular copper enzymes that initiate the cross-linking of collagens and elastin by catalyzing oxidative deamination of the ε-amino group in certain lysine and hydroxylysine residues. The cross-links formed are responsible for the tensile strength of collagen fibers, the unique elastic properties of elastin and overall ECM maturation. Four human lysyl oxidase (LOX) isoenzymes have been identified and characterized. The LOXL3 gene is expressed in several tissues, the highest expression levels being in the placenta, heart, ovary, testis, small intestine, and spleen. The LOXL4 gene is likewise expressed in most human tissues studied, the highest levels being seen in the skeletal muscle, testis, and pancreas. LOX proteins have been shown to be secreted extracellular proteins. LOX is critically required for the biosynthesis of functional extracellular matrices. However, excessive accumulation of insoluble collagen fibers also leads to fibrosis, and up-regulation of LOX expression and increased LOX activity is found in several fibrotic diseases. Development of specific inhibitors of LOX activity with the ultimate goal of preventing excessive formation of insoluble collagenous extracellular matrices is a potential therapeutic strategy to treat these fibrotic diseases. Lysyl oxidase is also known for its tumor suppressor activity and decreased levels of lysyl oxidase mRNA are found in most cancer cell lines as well as in murine cells transformed by several oncogenes. Few specific assay systems are available for LOX. Originally, a tritium-based assay was used to measure LOX activity. It involved the purification of LOX and tritium radio-labeled lysine or tropoelastin that then served as a substrate for LOX. This method does not work well to measure LOX activity in cell lysates or in animal tissues and homogenates. Recently several LOX assay systems have been developed utilizing the peroxide by-product of reaction. The reaction for lysyl oxidase proceeds as follows as it reacts with primary amines: RCH₂NH₂ + O₂ ^--à RCHO^- + NH₃ +_.Horseradish peroxidase with homovanillate and Amplex Red have both been used to try to measure the activity of LOX utilizing the H₂O₂ produced, but neither has proved to be reliable and reproducible. Marker Gene now provides several sensitive substrates that should be useful for fluorescence detection of LOX activity, including dihydrorhodamine 123 (M0545) (peroxide sensitive), biocytin hydrazide (M0128), fluorescein-5-semithiocarbazide (M01036) and NBD-hydrazide (M1025) (aldehyde labeling). For more information about these assays and techniques, please visit our website or check the references below. Palamakumbura AH, Trackman PC. (2002) “A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples.” Anal Biochem. 300(2): 245-51. X. Liu, Y. Zhao, J. Gao, B. Pawlyk, B. Starcher, J. A. Spencer, H. Yanagisawa, J. Zuo, T. Li, (2004) “Elastic fiber homeostasis requires lysyl oxidase−like 1 protein” Nature Genetics 36: 178-182. Wakasaki H., Ooshima A., (1990) “Immunohistochemical localization of lysyl oxidase with monoclonal antibodies.” Lab Invest. 63(3): 377-384. Kagan, H. M., Sullivan, K. A. (1982) “Lysyl oxidase: Preparation and role in elastin biosynthesis.” Methods Enzymol. 82: 637–650. Trackman, P. C., Zoski, C. G., Kagan, H. M. (1981) Development of a peroxidase-coupled fluorometric assay for lysyl oxidase. Anal. Biochem. 113: 336–342. Zhou, M., Diwu, Z., Panchuk-Voloshina, N., Haugland, R. P. (1997) A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: Applications in detecting the activity of phagocyte NADPH oxidase and other oxidases.” Anal. Biochem. 253: 162–168.
	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
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