Protocols and Applications Guide (US Letter Size) - Promega

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|||||||| 8Bioluminescence Reporters Synthetic poly(A) Selectable Marker – None – Hygror – Neor – Puror SV40 late poly(A) signal ori SV40 early enhancer/ promoter pGL4 Vectors Amp r Poly(A) block (for background reduction) Upstream Element – Multiple cloning region – Promoter/ response elements Luciferase Gene – Firefly (luc2) Rapid Response (–P, –CP) – Renilla (hRluc) Rapid Response (–P, –CP) Figure 8.5. The family of pGL4 Luciferase Reporter Vectors incorporates a variety of additional features, such as a choice of luciferase genes, Rapid Response versions, a variety of mammalian selectable markers, and vectors with or without promoters. sufficient or whether a greater density of information is desired. If a greater density of information is required, see the Dual-Reporter (and dual-color) Assays, Section III.B. Using an assay reagent that produces stable luminescence is more convenient when performing assays in multiwell plates. Unfortunately, because bright reactions fade relatively quickly, a trade-off is necessary between luminescence intensity and duration. The Bright-Glo Reagent is designed for firefly luciferase to yield maximal luminescence intensity and sufficient duration for analysis in a multiwell plate. The Steady-Glo® Reagent provides even greater luminescence duration but with lower intensity. Both reagents are designed to work directly in culture medium for mammalian cells, so prior cell lysis is not necessary. This allows the user to grow cells in multiwell plates and then measure expression with a single step. Additional Resources for Single-Reporter Assays Technical Bulletins and Manuals TB281 Luciferase Assay System Technical Bulletin (www.promega.com/tbs/tb281/tb281.html) TM055 Renilla Luciferase Assay System Technical Manual (www.promega.com /tbs/tm055/tm055.html) TM052 Bright-Glo Luciferase Assay System Technical Manual (www.promega.com /tbs/tm052/tm052.html) TM051 Steady-Glo® Luciferase Assay System Technical Manual (www.promega.com /tbs/tm051/tm051.html) Protocols & Applications Guide www.promega.com rev. 3/09 4897MA TM259 pGL4 Luciferase Reporter Vectors Technical Manual (www.promega.com /tbs/tm259/tm259.html) Citations Rifas, L. et al. (1997) Gestational exposure to ethanol suppresses msx2 expression in developing mouse embroyos. Proc. Natl. Acad. Sci. USA 94, 7549–54. Luciferase studies were performed on transfected MC3T3-E1 cell lysates using the Luciferase Assay System. Constructs were prepared in the pGL2 Promoter Vector. PubMed Number: 9207129 Ashfield , T. et al. (2004) Convergent evolution of disease resistance gene specificity in two flowering plant families. Plant Cell 16, 309–18. Leaves of Glycine max (soybean) were co-transfected by particle bombardment with various combinations of vectors encoding plant disease resistance genes and a luciferase reporter construct containing the constitutive 35S promoter of cauliflower mosaic virus. Leaf disks from the transfected areas were frozen in liquid nitrogen, ground and resuspended in 240μl of Luciferase Cell Culture Lysis Reagent. The lysates were then assayed for luciferase activity with the Luciferase Assay System. The luciferase values correlated to plant leaf cell survival of the various constructs. PubMed Number: 14742871 Citations de Haan, C.A.M. et al. (2004) Cleavage inhibition of the murine coronavirus spike protein by a furin-like enzyme affects cell-cell but not virus-cell fusion. J. Virol. 78, 6048–54. The Renilla Assay System was used to analyze mouse hepatitis coronavirus strain A59 (MHV-A59) entry into cells. A mouse hepatitis coronavirus construct expressing PROTOCOLS & APPLICATIONS GUIDE 8-8
|||||||| 8Bioluminescence Reporters Renilla luciferase was used to infect LR7 cells in the presence or absence of a furin protease inhibitor. PubMed Number: 15141003 Lin , P.F. et al. (2003) A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding. Proc. Natl. Acad. Sci. USA 100, 11013–8. To test the effect of BMS-378806, a new small molecule inhibitor of HIV-1, a cell fusion assay was developed. Target cells that stably expressed CD4, CXCR4 or CCRS receptors and carried a responsive luciferase plasmid were prepared. Effector cells were transiently transfected with HIV coat protein gp160 from various strains of virus, and a plasmid to activate the responsive element promoting luciferase. Therefore, if the cells fused, luciferase was activated. To measure the activation, effector cells were plated with target cells 1:2 and seeded into 96-well plates at 1 x 104 cells/well, then incubated with various concentrations of BMS-378806 for 12–24 hours. Luciferase activity was determined using Steady-Glo® Reagent. PubMed Number: 12930892 Promega Publications PN075 Bright-Glo and Steady-Glo®: Reagents for academic and industrial applications. (www.promega.com /pnotes/75/8554_03/8554_03.html) PN089 pGL4 Vectors: A new generation of luciferase reporter vectors. (www.promega.com /pnotes/89/12416_07/12416_07.html) B. Dual-Reporter Assays The most commonly used dual-reporter assay is based on combining the chemistries for firefly and Renilla luciferases. These luciferases use different substrates and thus can be differentiated by their enzymatic specificities. The method comprises adding two reagents to each sample, with a measurement of luminescence following each addition. Addition of the first reagent activates the firefly luciferase reaction; addition of the second reagent extinguishes firefly luciferase and initiates the Renilla luciferase reaction. The Dual-Luciferase® Assay Reagent relies on cell lysis prior to performing the assay and thus requires the use of reagent injectors if used with multiwell plates. The Dual-Glo Reagent is optimized for multiwell plates, providing longer luminescence duration (in other words, a longer luciferase half-life). As with other reagents designed for use in multiwell plates, the Dual-Glo Assay works directly in the culture medium for mammalian cells without the prior cell lysis. Generally the benefits of a dual assay can improve experimental efficiency by: i) reducing variability that can obscure meaningful correlations; ii) normalizing interfering phenomena that may be inherent in the experimental system; and iii) normalizing differences in transfection efficiencies between samples. Protocols & Applications Guide www.promega.com rev. 3/09 Reducing Variability Because cells are complex micro-environments, significant variability may occur between samples within an experiment and between experiments performed at different times. Challenges include trying to maintain uniform cell density and viability between samples and accomplishing reproducible transfection of exogenous DNA. Multiwell plates introduce variables such as edge effects, brought about by differences in heat capacity and humidity across a plate. Dual assays can control for much of this variability, leading to more accurate and meaningful comparisons between samples (Hawkins et al. 2002; Hannah et al. 1998; Wood, 1998; Faridi et al. 2003). Dual-Color Assays In some cases, researchers may prefer to activate both luciferase assays simultaneously by adding a single reagent. This reduces total assay volume and liquid handling requirements. The light emission of the two luciferases can be differentiated by the color of the luminescence. We have developed click beetle luciferases, which are related to firefly luciferase, to yield red and green luminescence. The structures of these luciferases are nearly identical, containing only a few amino acid substitutions necessary to create the different colors. This structural similarity means that both the control reporter and the experimental reporter are likely to respond similarly to biochemical changes within the cell, resulting in even more accurate normalization to the control. The genes encoding these reporters, the Chroma-Luc genes, are codon optimized for mammalian cells. We developed two genes encoding the green-emitting reporter, one which is nearly identical to the gene encoding red luminescence, and one that is maximally divergent from it but that encodes the same protein. The divergent gene may be useful under circumstances where genetic recombination is a concern. The Chroma-Glo Assay reagent is designed for use in multiwell plates. Its formulation supports optimal reaction kinetics for both reporters simultaneously, and it works directly in culture medium. Because color differentiation is required for the Chroma-Glo Assay, a luminometer capable of using colored optical filters is required. Since the light is transmitted through the optical filters, sensitivity relative to other assay methods is reduced. Both luciferases may be detectable using optical filters when the relative concentrations differ by approximately 100-fold. This is less than dual-luciferase assays using chemical differentiation, where the relative concentration may differ by over 1,000-fold. Distinguishing among the Dual Assays Dual-reporter and dual-color assays allow the user either to measure expression of two different reporter genes or one reporter gene and cell viability. In all cases, the assays allow both measurements to be made sequentially from each sample. Most dual assays are optimized for use in multiwell plates. PROTOCOLS & APPLICATIONS GUIDE 8-9
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CONTENTS I. Nucleic Acid Amplificat
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| 1Nucleic Acid Amplification CONTE
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| 1Nucleic Acid Amplification Unamp
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| 1Nucleic Acid Amplification Capoz
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| 1Nucleic Acid Amplification One i
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| 1Nucleic Acid Amplification comig
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| 1Nucleic Acid Amplification inclu
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| 1Nucleic Acid Amplification polym
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| 1Nucleic Acid Amplification Addit
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| 1Nucleic Acid Amplification 3. Pl
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| 1Nucleic Acid Amplification 13. S
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| 1Nucleic Acid Amplification IX. R
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| 1Nucleic Acid Amplification Hoppe
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|| 2RNA Interference CONTENTS I. RN
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|| 2RNA Interference VII. Reference
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|| 2RNA Interference Wianny, F. and
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||| 3Apoptosis I. Introduction A. C
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||||| 5Protein Expression I. Introd
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||||||||||||| 13Cloning CONTENTS I.
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