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Bioluminescence Resonance Energy Transfer (BRET)
High resolution imaging of luciferase activity at the single cell level has been problematic for such techniques as FACS or high-field fluorescent microscopic analysis. Recently, several methods have been developed based upon luciferase-GFP (green fluorescent protein) methods that have approached this problem from a new angle. Many luminous marine organisisms use a method call BRET (Bioluminescent Resonance Energy Transfer) that is very similar to FRET in which the light emission from a luciferase-luciferin reaction is used to induce a GFP fluorescence, that can be longer lived and measured at the single cell level. The BRET technique has also found application to protein-protein interaction studies, since fusion proteins linked to luciferase and GFP exhibit a resonance energy transfer radius of about 10 nm. In order to create a suitable overlap for the luciferase light emission to activate standard GFP's, a coelenterazine derivative, bisdeoxycoelenterazine (also called DeepBlue C) has often been used as the substrate. Recently, however, work from the laboratory of Dr. Hideto Hoshino and collaborators at the Department of Photobiology at Hokkaido University in Japan, have been able to utilize standard coelenterazine as a substrate for renilla luciferase in combination with a YFP (Yellow Fluorescent Protein) pair for BRET that has a much higher light output. Expression vectors containing the YFP and Renilla reniformis genes have been developed and have shown improved spacial and temporal resolution for light emission in living cells, although a CCD camera using a 10 second exposure was still necessary for single cell analysis. For more information about these new systems, please visit our website or see the references below.

• Welsh DK, Yoo SH, Liu AC, Takahashi JS, Kay SA (2004) "Bioluminescence imaging of individual fibroblasts reveals persistent, independently phased circadian rhythms of clock gene expression." Curr. Biol.14(24): 2289-95.
• Welsh DK, Imaizumi T, Kay SA (2005) "Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and Mammalian cells." Methods Enzymol. 393: 269-88.
• Bertrand L, Parent S, Caron M, Legault M, Joly E, Angers S, Bouvier M, Brown M, Houle B, Ménard L (2002) "The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS)." J. Recept. Signal Transduct. Res. 22(1-4): 533-41.
• Jensen AA, Hansen JL, Sheikh SP, Bräuner-Osborne H, (2002) "Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)." Eur. J. Biochem. 269(20): 5076-87.
• Nakamura, H., Wu, C., Murai, A., Inouye, S. (1997) "Efficient bioluminescence of bisdeoxycoelenterazine with the luciferase of a deep-sea shrimp Oplophorus." Tet. Lett. 38(36): 6405-6406.
• De, A, Gambhir, SS (2005) "Noninvasive imaging of protein-protein interactions from live cells and living subjects using bioluminescence resonance energy transfer." FASEB Journal 19(14): 2017.

GFP Blinking used as a Molecular Thermometer.

• Wong, FHC, Banks, DS, Abu-Arish, A, Fradin, C, (2007) "A Molecular Thermometer Based on Fluorescent Protein Blinking" J. Amer. Chem. Soc. 129:10302-10303.
• Boas, G.,(2007) "Blinking Yields Improved Molecular Thermometer" 14(9): 12.
• Karstens, T., and K. Kobs (1980) "Rhodamine B and rhodamine 101 as reference substances for fluorescence quantum yield measurements." J. Phys. Chem. 84: 1871-1872.
• Haupts, U, Maiti, S, Schwille, P, Webb WW, (1998) "Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation Spectroscopy." Proc. Natl. Acad. Sci. USA. 95:13573-13578.

Amylase Determination Using Long-Wavelength Detection.
α-Amylase (EC 3.2.1.1) is an important endo-glycosidase that hydrolyzes α-1,4 glycosidic linkages of D-glucose oligomers and polymers. It is a key enzyme of carbohydrate metabolism in mammals, plants, and bacteria. Measurement of α-amylase activity is very important for the diagnosis of pancreatic and salivary diseases as well as in the food industry. The measurement of α-amylase (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1) is the most widely used test for diagnosing acute pancreatitis. Many assay methods for α-amylase activity have been reported in the literature, many of which use simple maltooligosaccharide derivatives attached to a chromophore or fluorophore as the aglycone. However, these assays can require the combined use of auxiliary enzymes, such as added α-glucosidase or glucoamylase, in order to generate the chromophore or fluorophore from the glycosides. Several other systems have been developed based upon heavily labeled cyclodextrins or starches. To date, there are very few assays that directly and simply measure the hydrolysis of maltooligosaccharide derivatives spectroscopically. Marker Gene is developing a new long-wavelength fluorescent α-amylase assay that can be used to directly measure α-amylase activity in a continuous assay format directly from biological samples. This assay will be available soon, and you can obtain more information about the assay and method by writing us at techservice@markergene.com or by calling our technical assistance staff toll-free at 1-888-218-4062. Please also see the references below for more information about α-amylase and its assay .

• Morishita, Y. Iinuma, Y, Nakashima, N, Majima, K, Mizoguchi K, . Kawamura, Y, (2000) Clin. Chem. 46: 928.
• Nishimura, S, Kimura, N, Matsuoka K, Lee, YC, Carbohydr. Lett. 4 (2001), p. 77.
• Murayama, T, Tanabe, T, Ikeda, H, Ueno A, (2006) "Direct assay for α-amylase using fluorophore-modified cyclodextrins." Bioorganic & Medicinal Chemistry 14(11): 3691-3696.
• Lorentz, K (2000) "Routine α-Amylase Assay Using Protected 4-Nitrophenyl-1,4-α-D-maltoheptaoside and a Novel α-Glucosidase." Clinical Chemistry 46: 644-649.
• Ogawa, K, Matsui, H, Usui, T, (1992). "Differential assay of human pancreatic and salivary alphaamylases with p-nitrophenyl 65-o-beta-D-galacopyraosyl-alpha-maltopentaoside as the subtrate" Biosci. Biotech. Biochem. 56: 1933–1936.
  

Fluorescent Protease Assays.
Direct fluorescence-based assays for detecting metallo-, serine, acid or sulfhydryl proteases are important in medical, biochemical and cell biology research. Analysis of low levels of protease activity is important in biochemical quality-control testing, for analysis of protease inhibitors or cofactors, as well as for basic research application in biology and molecular biology. Several fluorescence-based methods have been developed for detecting protease activity including the fluorescein thiocarbamoyl (FTC)-casein protease assay, in which unhydrolyzed protein must be precipitated with trichloroacetic acid, separated by centrifugation, transferred for measurement and then pH-adjusted to optimize the fluorescence signal. Several methods take advantage of the self-quenching of fluorescein or other dyes when heavily coupled to proteins such as casein or BSA. These methods do not involve separation steps and are up to 100 times more sensitive than the FTC-casein assay. Such casein conjugates that are labeled with multiple fluorescent dyes, exhibit almost total fluorescence quenching. Protease-catalyzed hydrolysis releases highly fluorescent-labeled peptides, which are then read in a continuous assay format. The accompanying increase in fluorescence is proportional to protease activity and can be conveniently measured using a fluorometer equipped with an appropriate (fluorescein) filter set. In addition to utility for detecting protease contamination of culture media and other experimental samples, the assay can be used to continuously measure the kinetics of a variety of exo- and endopeptidases or to measure the total substrate turnover at a fixed time following addition of the enzyme. Among the enzymes that can be monitored using this method are elastase, chymotrypsin, thermolysin, trypsin, papain, pepsin, cathepsin D and elastase. Other methods that have been utilized to measure protease activity include fluorescence polarization measurements or ethidium bromide binding to DNA after protease digestion of histones. For more information about these methods, please see the references below, or visit our website.

• Anson, M.L., (1938) "The estimation of pepsin, trypsin, papain and cathepsin with hemoglobin" J. Gen. Physiol. 22: 79-89.
• Severini, A , Morgan, AR, (1991) "An assay for proteinases and their inhibitors based on DNA/ethidium bromide fluorescence." Anal. Biochem. 193: 83.
• Folin, O., Ciocalteu, V., "On tyrosine. and tryptophane determinations in proteins."(1929) J. Biol. Chem. 73, 627
• Twining SS, (1984) "Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes." Anal. Biochem. 143: 30-34.
• Homer KA, Beighton D. (1990) "Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates." Anal Biochem. 191(1):133–137.
• Voss, EW, Workman,CJ, Mummert ME, (1996) "Detection of Protease Activity Using a Fluorescence-Enhancment Globular Substrate" BioTechniques 20(2): 286-291.
• Bolger R, Checovich W, (1994) "A New Protease Activity Assay Using Fluorescence Polarization" BioTechniques 17(3): 585-589.

• 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.

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.