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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 website or visit one of our distributors:      Marker Gene Monthly Newsletter October, 2007 Volume 7, 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. HSV1-TK Marker Gene Assays. In order to confront these problems, several assays have been developed for measuring TK activity in transfected cells. Among the most common is the use of Immunofluorescence staining or radioactively labeled nucleosides. Recently the fluorescently labeled uridine derivative 5-amino-2-deoxyuridine-dansyl chloride (AUdR-DANS) has been prepared as a direct cell stain for TK. Coupling of 5-dimethylamino-1-naphthalene-sulfonyl-chloride (dansyl chloride) to the 5 position of 5-amino-2-deoxyuridine provides a fluorescent nucleotide analog (AUdR-DANS) that is a substrate for the TK enzyme, and can be used in cellular assays of thymidine kinase. Exponentially growing monolayers of adherent cells can be treated with AUdR -DANS (5uM for one hour), washed with PBS and visualized by excitation at 420 nm, with emission at 500 nm. The phosphorylated nucleotide analog is well retained inside transfected cells and can be visualized by microscopy or quantitated by microplate assay techniques. For more information about these methods, please visit our website or see the references below.
 New Fluorescent Protease Assay Kit. 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. The MarkerGeneTM Fluorescent Protease Assay Kit avoids these time-consuming separation steps by taking advantage of the self-quenching of fluorescein when heavily coupled to protein. This kit uses the conjugated protein, FITC-Casein (Product M1315) as a substrate. Casein is a naturally occurring protein in milk that is suitable as a general substrate for a myriad of proteases. Labeled with multiple fluorescent dyes, the substrate exhibits significant fluorescence quenching. Protease-catalyzed hydrolysis releases highly fluorescent-labeled peptides; the accompanying increase in fluorescence is proportional to protease activity and can be conveniently measured in a continuous assay format using a fluorometer equipped with an appropriate (fluorescein) filter set (EX/EM= 490/520 nm). This kit has demonstrated sensitivity of less than 1mU/mL enzyme. Extensive protease cleavage of the substrate can result in fluorescence increases of greater than 10-fold. 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. For more information about these new assays and methods, please see the references below or visit our website.
Chloramphenicol Acetyl Transferase (CAT) Assay. To perform the enzyme assay, cells are lysed and the extracted proteins are mixed with a chloramphenicol analog that has either been synthesized with a fluorescent or radioactive group. In addition, the substrate acetyl CoA is also added to the mixture. The amount of acetylation measured is then directly proportional to the amount of CAT enzyme expressed in the original cell lysate sample. Therefore, you can measure the amount of acetylated chloramphenicol in different cell or tissue extracts and determine how much protein was produced as a result of activated promoters or other elements in vivo. There are two hydroxyl groups on chloramphenicol, and although the CAT enzyme adds acetate only to the primary hydroxyl, it can equilibrate with the secondary -OH, leading to either one or two acetyl groups added to each chloramphenicol. When the CAT reaction is completed, the products are placed on thin-layer chromatographic (TLC) sheets, placed in the appropriate solvent (a mixture of chloroform and methanol (95:5)) and allowed to migrate on the TLC sheet which is then visuallized by UV absorption or exposed to X-ray film. Each product (two forms of chloramphenicol with one acetyl group and one with two acetyl groups added) and all the unused substrate will migrate on the TLC surface according to their structures. Since the only labeled molecules are the chloramphenicol or its analogs, these will be the only molecules visible on the TLC plate. CAT assays offer an indirect but quantitative way to measure the amount of transcription driven from any given expression system where the marker gene is active. For more information about these assays, please see the references below or visit our website.
The lower retention of carboxyfluorescein provides highly fluorescent live cell staining, while the transition to the PI positive cell is preceded by a lowering in CF fluorescence due to leakage from damaged or dead cells. It is possible to use the succinimidyl ester of CFDA (5(6)-Carboxyfluorescein, NHS ester, CFDA-SE, M0013) to achieve permanently bound green fluorescence and permanently label all target cells. This is useful for detecting both live and dead cells using the green fluorescence line, for the total cell count. But this combination can also be used to investigate the impact of cell preparation on cell integrity. CFDA-SE stained cells that remained PI negative indicate integrity (green color), while cells destroyed in the preparation will be double positive for both, CF and PI. Cells dead prior to the cell preparation were CF negative but stained for PI positively (red). The influence of cell preparation can be important for reliable cytotoxicity assay development, especially where aggregation of cells and adhesion of cell debris to otherwise intact cells is found. For more information about these techniques, please visit our website or see the references below.
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 http://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 at 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:
  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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Cytotoxicity Testing.