Dual_Luciferase_Reporter_Assay_System_Protocol

Te c h n i c a l M a n u a l IN TE G R A TE D SO LU T IO N S world-c lass SERVICE & SUPPORT IN TE G R A TE D SO LU T IO N S use me with GLOMAX ® INSTRUMENTS Dual-Luciferase® Reporter Assay System INSTRUCTIONS FOR USE OF PRODUCTS E1910 AND E1960. PRINTED IN USA. Revised 6/11 Part# TM040 tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page a Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM040 Revised 6/11 Page 1 1. Description ..........................................................................................................2 A. Dual-Luciferase® Reporter Assay Chemistry...................................................3 B. Format of the Dual-Luciferase® Reporter Assay .............................................5 C. Passive Lysis Buffer .............................................................................................6 2. Product Components and Storage Conditions ............................................8 3. The pGL4 Luciferase Reporter Vectors .........................................................9 A. Description of pGL4 Vectors ..............................................................................9 B. Important Considerations for Co-Transfection Experiments ........................9 4. Instrument Considerations ............................................................................10 A. Single-Sample Luminometers...........................................................................10 B. Multi-Sample and Plate-Reading Luminometers..........................................10 C. Scintillation Counters.........................................................................................11 5. Preparation of Cell Lysates Using Passive Lysis Buffer..........................12 A. Passive Lysis Buffer Preparation .....................................................................12 B. Passive Lysis of Cells Cultured in Multiwell Plates .....................................12 C. Active Lysis of Cells by Scraping ....................................................................13 6. Dual-Luciferase® Reporter Assay Protocol.................................................14 A. Preparation of Luciferase Assay Reagent II ...................................................14 B. Preparation of Stop & Glo® Reagent ...............................................................15 C. Standard Protocol ...............................................................................................15 D. Important Considerations for Cleaning Reagent Injectors ..........................18 E. Determination of Assay Backgrounds ............................................................19 7. References .........................................................................................................21 8. Appendix ...........................................................................................................22 A. Composition of Buffers and Solutions ............................................................22 B. Related Products.................................................................................................22 Dual-Luciferase® Reporter Assay System All technical literature is available on the Internet at: www.promega.com/tbs/ Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail: techserv@promega.com. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 1 1. Description Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology. Applications include the study of receptor activity, transcription factors, intracellular signaling, mRNA processing and protein folding. Dual reporters are commonly used to improve experimental accuracy. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system. Typically, the “experimental” reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by differences in cell viability or transfection efficiency. Other sources of variability, such as differences in pipetting volumes, cell lysis efficiency and assay efficiency, can be effectively eliminated. Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing extraneous influences. The Dual-Luciferase® Reporter (DLR™) Assay System(a–f) provides an efficient means of performing dual-reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases are measured sequentially from a single sample. The firefly luciferase reporter is measured first by adding Luciferase Assay Reagent II (LAR II) to generate a stabilized luminescent signal. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is simultaneously initiated by adding Stop & Glo® Reagent to the same tube. The Stop & Glo® Reagent also produces a stabilized signal from the Renilla luciferase, which decays slowly over the course of the measurement. In the DLR™ Assay System, both reporters yield linear assays with subattomole sensitivities and no endogenous activity of either reporter in the experimental host cells. Furthermore, the integrated format of the DLR™ Assay provides rapid quantitation of both reporters either in transfected cells or in cell-free transcription/translation reactions. Promega offers the pGL4 series of firefly and Renilla luciferase vectors designed for use with the DLR™ Assay Systems. These vectors may be used to co-transfect mammalian cells with experimental and control reporter genes. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM040 Printed in USA. Page 2 Revised 6/11 tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 2 1.A. Dual-Luciferase® Reporter Assay Chemistry Firefly and Renilla luciferases, because of their distinct evolutionary origins, have dissimilar enzyme structures and substrate requirements. These differences make it possible to selectively discriminate between their respective bioluminescent reactions. Thus, using the DLR™ Assay System, the luminescence from the firefly luciferase reaction may be quenched while simultaneously activating the luminescent reaction of Renilla luciferase. Firefly luciferase is a 61kDa monomeric protein that does not require post- translational processing for enzymatic activity (1,2). Thus, it functions as a genetic reporter immediately upon translation. Photon emission is achieved through oxidation of beetle luciferin in a reaction that requires ATP, Mg2+ and O2 (Figure 1). Under conventional reaction conditions, the oxidation occurs through a luciferyl-AMP intermediate that turns over very slowly. As a result, this assay chemistry generates a “flash” of light that rapidly decays after the substrate and enzyme are mixed. Many of our Luciferase Assay Reagents for quantitating firefly luciferase incorporate coenzyme A (CoA) to provide more favorable overall reaction kinetics (3). In the presence of CoA, the luciferase assay yields stabilized luminescence signals with significantly greater intensities (Figure 2) than those obtained from the conventional assay chemistry. The firefly luciferase assay is extremely sensitive and extends over a linear range covering at least seven orders of magnitude in enzyme concentration (Figure 3). Renilla luciferase, a 36kDa monomeric protein, is composed of 3% carbohydrate when purified from its natural source, Renilla reniformis (4). However, like firefly luciferase, post-translational modification is not required for its activity, and the enzyme may function as a genetic reporter immediately following translation. The luminescent reaction catalyzed by Renilla luciferase utilizes O2 and coelenterate-luciferin (coelenterazine; Figure 1). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM040 Revised 6/11 Page 3 Figure 1. Bioluminescent reactions catalyzed by firefly and Renilla luciferases. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 3 1.A. Dual-Luciferase® Reporter Assay Chemistry (continued) In the DLR™ Assay chemistry, the kinetics of the Renilla luciferase reaction provide a stabilized luminescent signal that decays slowly over the course of the measurement (Figure 2). Similar to firefly luciferase, the luminescent reaction catalyzed by Renilla luciferase also provides extreme sensitivity and a linear range generally extending six orders of magnitude (Figure 3). Note that the effective range of the luminescent reactions may vary depending on the type of luminometer (e.g., 96-well versus single-sample) used. An inherent property of coelenterazine is that it emits low-level autoluminescence in aqueous solutions. Originally this drawback prevented sensitive determinations at the lower end of enzyme concentration. Additionally, some types of nonionic detergents commonly used to prepare cell lysates (e.g., Triton® X-100) greatly intensify coelenterazine autoluminescence. The DLR™ Assay Systems include proprietary chemistry that reduces autoluminescence to a level that is not measurable for all but the most sensitive luminometers. Passive Lysis Buffer is formulated to minimize the effect of lysate composition on coelenterazine autoluminescence. In addition, the DLR™ Assay Systems include two reconstituted assay reagents, Luciferase Assay Reagent II and Stop & Glo® Reagent, that combine to suppress coelenterazine autoluminescence. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM040 Printed in USA. Page 4 Revised 6/11 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 Ac tiv ity (% p ea k) Time (sec) Firefly Renilla Figure 2. Luminescent signals generated in the Dual-Luciferase® Reporter Assay System by firefly and Renilla luciferases. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 4 iamlucyliu 高亮 1.B. Format of the Dual-Luciferase® Reporter Assay Quantitation of luminescent signal from each of the luciferase reporter enzymes may be performed immediately following lysate preparation without the need for dividing samples or performing additional treatments. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to Luciferase Assay Reagent II. Quenching of firefly luciferase luminescence and concomitant activation of Renilla luciferase are accomplished by adding Stop & Glo® Reagent to the sample tube immediately after quantitation of the firefly luciferase reaction. The luminescent signal from the firefly reaction is quenched by at least a factor of 105 (to ≤0.001% residual light output) within 1 second following the addition of Stop & Glo® Reagent (Figure 4). Complete activation of Renilla luciferase is also achieved within this 1-second period. When using a manual luminometer, the time required to quantitate both luciferase reporter activities will be approximately 30 seconds. The procedure can be summarized as follows: Elapsed Time Step 1: Manually add prepared lysate to Luciferase Assay ~3 seconds Reagent II predispensed into luminometer tubes; mix. Step 2: Quantify firefly luciferase activity. 12 seconds Step 3: Add Stop & Glo® Reagent; mix. 3 seconds Step 4: Quantitate Renilla luciferase activity. 12 seconds Total elapsed time for the DLR™ Assay 30 seconds Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM040 Revised 6/11 Page 5 10 04 2M A Firefly luciferase Renilla luciferase r² = 0.9996 r² = 0.9993 1 × 103 1 × 104 1 × 105 1 × 106 1 × 107 1 × 108 1 × 109 1 × 1010 Luciferase Concentration (moles/reaction) 1 × 10 –2 0 1 × 10 –1 9 1 × 10 –1 8 1 × 10 –1 7 1 × 10 –1 6 1 × 10 –1 5 1 × 10 –1 4 1 × 10 –1 3 1 × 10 –1 2 Figure 3. Comparison of the linear ranges of firefly and Renilla luciferases. The DLR™ Assay was performed with a mixture of purified firefly and Renilla luciferases prepared in PLB containing 1mg/ml BSA. A Promega GloMax® 20/20 Luminometer was used to measure luminescence. As shown in this graph with the DLR™ Assay System, the linear range of the firefly luciferase assay is eight orders of magnitude, providing detection sensitivity of ≤0.1 femtogram (approximately 10–21mole) of firefly luciferase reporter enzyme. The Renilla luciferase assay has a linear range covering eight orders of magnitude and allows for the detection of approximately 0.1 femtogram (approximately 10–21mole) of Renilla luciferase. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 5 iamlucyliu 高亮 iamlucyliu 高亮 1.B. Format of the Dual-Luciferase® Reporter Assay (continued) 1.C. Passive Lysis Buffer Passive Lysis Buffer (PLB) is specifically formulated to promote rapid lysis of cultured mammalian cells without the need to scrape adherent cells or perform additional freeze-thaw cycles (active lysis). Furthermore, PLB prevents sample foaming, making it ideally suited for high-throughput applications in which arrays of treated cells are cultured in multiwell plates, processed into lysates and assayed using automated systems. Although PLB is formulated for passive lysis applications, its robust lytic performance is of equal benefit when harvesting adherent cells cultured in standard dishes using active lysis. Regardless of the preferred lysis method, the release of firefly and Renilla luciferase reporter enzymes into the cell lysate is both quantitative and reliable for cultured mammalian cells (Figure 5). Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM040 Printed in USA. Page 6 Revised 6/11 1,000,000 100,000 10,000 1,000 100 1 0.10 10 Firefly Luciferase Activity Renilla Luciferase Activity Quenched Reporter #1 Luminescence 80,600 0.28 116,800 Reporter #1 Reporter #2 0.0004% Residual Activity Lu m in es ce nc e (R LU ) Figure 4. Measurement of luciferase activities before and after the addition of Stop & Glo® Reagent. The DLR™ Assay allows sequential measurement of firefly luciferase (Reporter #1), followed by Renilla luciferase activity (Reporter #2) on addition of Stop & Glo® Reagent to the reaction. Both reporter activities were quantitated within the same sample of lysate prepared from CHO cells co-transfected with pGL3 Control Vector (Cat.# E1741) and pRL-SV40 Vector (Cat.# E2231). To demonstrate the efficient quenching of Reporter #1 by Stop & Glo® Reagent, an equal volume of Stop & Glo® Buffer (which does not contain the substrate for Renilla luciferase) was added. Firefly luciferase luminescence was quenched by greater than 5 orders of magnitude. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 6 In addition to its lytic properties, PLB is designed to provide optimum performance and stability of the firefly and Renilla luciferase reporter enzymes. An important feature of PLB is that, unlike other cell lysis reagents, it elicits only minimal coelenterazine autoluminescence. Hence, PLB is the lytic reagent of choice when processing cells for quantitation of firefly and Renilla luciferase activities using the DLR™ Assay System. Other lysis buffers (e.g., Glo Lysis Buffer, Cell Culture Lysis Reagent and Reporter Lysis Buffer) either increase background luminescence substantially or are inadequate for passive lysis. If desired, the protein content of cell lysates prepared with PLB may be readily quantitated using a variety of common chemical assay methods. Determination of protein content must be performed using adequate controls. Diluting lysates with either water or a buffer that is free of detergents or reducing agents is recommended in order to reduce the effects that Passive Lysis Buffer may have on background absorbance. A standard curve with BSA must be generated in parallel under the same buffer conditions. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM040 Revised 6/11 Page 7 Passive Lysis Active Lysis 120 110 100 90 80 70 60 50 40 30 20 10 0 % F ire fly L uc ife ra se A ct iv ity Lysis Method CHO CV-1 HeLa NIH/3T3 A. Firefly Luciferase Assay 120 110 100 90 80 70 60 50 40 30 20 10 0 % R en ill a Lu ci fe ra se A ct iv ity Lysis Method CHO CV-1 HeLa NIH/3T3 B. Renilla Luciferase Assay 14 03 M A0 3_ 6A Figure 5. Comparison of firefly and Renilla luciferase reporter activities in cell lysates prepared with Passive Lysis Buffer using either the passive or active lysis procedure. Four different mammalian cell types were co-transfected with firefly and Renilla luciferase expression vectors. Lysates were prepared by either exposing adherent cells to Passive Lysis Buffer for 15 minutes (passive lysis), or scraping adherent cells in the presence of Passive Lysis Buffer followed by one freeze-thaw cycle (active lysis). For comparative purposes, reporter activities were normalized to those obtained with the active lysis method for each cell type. tm040.0610:EIVD_TM.qxd 6/17/2011 9:38 AM Page 7 2. Product Components and Storage Conditions Product Size Cat.# Dual-Luciferase® Reporter Assay System 100 assays E1910 Each system contains sufficient reagents to perform 100 standard Dual-Luciferase® Reporter Assays. Includes: • 10ml Luciferase Assay Buffer II • 1 vial Luciferase Assay Substrate (Lyophilized Product) • 10ml Stop & Glo® Buffer • 200µl Stop & Glo® Substrate, 50X • 30ml Passive Lysis Buffer, 5X Product Size Cat.# Dual-Luciferase® Reporter Assay System, 10-Pack 1,000 assays E1960 Each system contains sufficient reagents to perform 1,000 standard Dual-Luciferase® Reporter Assays using 96-well luminometry plates. Includes: • 10 × 10ml Luciferase Assay Buffer II • 10 × 1 vial Luciferase Assay Substrate (Lyophilized Product) • 10 × 10ml Stop & Glo® Buffer • 10 × 200µl Stop & Glo® Substrate, 50X • 30ml Passive Lysis Buffer, 5X Note regarding Cat.# E1960: For applications requiring more lysis reagent (e.g., >100µl/well), additional Passive Lysis Buffer may be purchased separately (Cat.# E1941). Storage Conditions: Upon receipt, store the Dual-Luciferase® Reporter Assay System at –20°C. Once the Luciferase Assay Substrate has been reconstituted, it should be divided into working aliquots and stored at –20°C for up to 1 month or at –70°C for up to 1 year. Ideally, Stop & Glo® Reagent (Substrate + Buffer) should be prepared just before each use. If necessary, this reagent may be stored at –20°C for 15 days with no decrease in activity. If stored at 22°C for 48 hours, the reagent’s activity decreases by 8%, and if stored at 4°C for 15 days, the reagent’s activity decreases by 13%. The Stop & Glo® Reagent can be thawed at room temperature up to 6 times with ≤15% decrease in activity. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-95