Benefits • Highsensitivity quantitation of luciferase activity with 6decade linear range • Automatic data analysis and calculation of normalized reporter activity in SoftMax Pro Software • SmartInject™ Technology for reagent mixing with optimal results DualLuciferase Reporter (DLR) Assay on the SpectraMax i3x MultiMode Microplate Reader with SpectraMax Injector Cartridge Introduction Reporter gene assays are used to study the expression of eukaryotic genes In dual reporter gene assays cells are transfected with two vectors the first containing an experimental reporter gene coupled to a regulated promoter of interest and the second containing a control reporter gene coupled to a constitutive promoter Normalizing the activity of the experimental reporter to the control reporter minimizes experimental variability Bioluminescent reporter systems using firefly and Renilla luciferases are widely used as coreporters because both assays are easy to run and exquisitely sensitive Promega’s DualLuciferase® Reporter (DLR) Assay System allows users to measure both firefly and Renilla luciferase activity in a single microplate well with firefly acting as the experimental reporter and Renilla the control Figure 1 illustrates the two enzymatic reactions which take place sequentially within the same assay well Firefly luciferase enzyme catalyzes the oxidation of luciferin with the concomitant release of light1 The reaction requires ATP Mg2+ and O2 Renilla luciferase catalyzes the O2dependent oxidation of coelenterate luciferin (coelenterazine) but does not require Mg2+ or ATP2 The enzymes have different substrate requirements so they can both be measured in a single reaction mixture The DLR assay requires delivery of two separate reagents containing the different substrates each followed by a luminescence read This assay workflow is easily performed using the SpectraMax i3x MultiMode Microplate Reader with SpectraMax Injector Cartridge which is fully DLReady validated3 In this application note we demonstrate a linear dynamic APPLICATION NOTE range of 6 decades for recombinant firefly and Renilla luciferases as well as linear detection of luciferasetransfected cells from 195 to 25000 cells per well Materials • DualLuciferase Reporter Assay System (Promega cat #E1960) contents include • Luciferase Assay Buffer II • Luciferase Assay Substrate • Stop & Glo Buffer • Stop & Glo Substrate • 5X Passive Lysis Buffer • Purified recombinant luciferase enzymes • Firefly luciferase QuantiLum® Recombinant Luciferase (Promega cat #E1701) • Renilla luciferase Recombinant Renilla Luciferase (RayBiotech cat # RB150003P10) • CHOK1 cells (ATCC cat #CCL61) Figure 1 Reactions catalyzed by firefly and Renilla luciferases Firefly and Renilla luciferase have different substrate requirements luciferin + ATP + O2 + O2 + AMP + PPi+ CO2 + hv Mg2+ HO COH firefly luciferase N S N S coelenterazine HO O OH N H N N OO N S N S Renilla luciferase HO O OH N N + CO2 + hv + light + light • Control luciferase vectors • pGL413[luc2SV40] firefly luciferase vector (Promega cat #E6681) • pGL474[hRlucTK] Renilla luciferase vector (Promega cat #E6921) • Fugene HD Transfection Reagent (Promega cat #E2311) • 6well tissue culture plates (Corning cat #3516) • 96well flat clear bottom white TCtreated microplates (Corning cat #3610) • BrightMax sealing films (Genesee cat #12639) • White 96 and 384well microplates (Greiner cat #655075 and #781075) • SpectraMax i3x MultiMode Microplate Reader • SpectraMax Injector Cartridge Methods Enzyme standard curves A stock solution of firefly luciferase was prepared by diluting the 124 mgmL solution provided to 1 mgmL with 1X Passive Lysis Buffer (PLB a component of the DualLuciferase Reporter Assay System) containing 1 mgmL BSA Stock Renilla luciferase was prepared by reconstituting the lyophilized enzyme with 1X PBS to a concentration of 1 mgmL Working solutions (10 µgmL) of firefly and Renilla luciferases were made by transferring 10 µL of stock solution (1 mgmL) into 990 µL PLB A combined luciferase stock standard (100 ngmL each) was then prepared by transferring 10 µL of each luciferase working solution into 980 µL PLB Serial 110 dilutions of the combined stock standard yielded standards with concentrations ranging from 100 fgmL to 100 ngmL (16 fM to 16 nM) Luciferase Assay Reagent II (LAR II) and Stop & Glo Reagent were prepared according to the DualLuciferase Reporter Assay System technical manual Injector 1 of the SpectraMax Injector Cartridge was primed with 260 µL of LAR II and Injector 2 was primed with 260 µL of Stop & Glo Reagent In SoftMax Pro the Acquisition View was used to configure the plate read with injection (Figure 2) Both injectors were set to deliver 100 µL (96well plate) or 25 µL (384well plate) of reagent using SmartInject Technology which combines injection with plate shaking for Figure 2 Acquisition Plan for DLR The Acquisition Plan editor in SoftMax Pro enables a dragand drop setup of operations to be applied to each well Shown above is the setup for the DLR assay Two separate inject and read steps were applied SmartInject depicted in the graphic above the sample Acquisition Plan enables shaking during the injection and 2second delay step so that reagents are mixed thoroughly and signal develops quickly and consistently from well to well Dispense and Shake Read Figure 3 96 and 384well dualluciferase standard curves A 10 dilution series of purified recombinant firefly (red plot) and Renilla (blue plot) luciferases was assayed using the DLR system A linear range of 6 decades was measured (r2 0994) Top 96well format bottom 384well format Luciferase (M) RLU Luciferase (M) RLU 96well plate 384well plate complete reagent mixing followed by a 2second delay and 10second integration 20 µL (96well plates) or 10 µL (384well plates) of each luciferase standard was pipetted into assay wells The assay plate was placed in the plate drawer of the SpectraMax i3x reader and the read was initiated Data analysis and graphing were performed using SoftMax Pro Software A preconfigured DualLuciferase Reporter Assay protocol is included in the SoftMax Pro protocol library Cellbased assay CHOK1 cells were plated at 25x105 cells per well in 6well culture plates and allowed to attach and grow overnight The next day cells were transiently transfected with pGL413[luc2SV40] firefly luciferase vector and pGL474[hRlucTK] Renilla luciferase vector following a standard protocol for the Fugene HD transfection reagent A ratio of 101 fireflyRenilla vector DNA was used and each well received a total of 1 µg DNA and 3 µL Fugene HD transfection reagent 24 hours after transfection cells were seeded in a 96well flat clear bottom white TCtreated microplate at densities from 195 to 25000 cells per well and allowed to grow overnight They were then lysed with 1X Passive Lysis Buffer and assayed in the same plate using the DualLuciferase Reporter Assay System and SpectraMax i3x reader with injector cartridge as described above Prior to assay a solid white plate seal was applied to the bottom of the microplate to maximize detection of luminescence signal Results Luciferase standard curves Signal for firefly and Renilla luciferase was linear across the 6decade range of enzyme tested from 16 fM to 16 nM (Figure 3) For the 96well plate this equates to 1 fg per well to 1 ng per well for the 384well plate it is 05 fg per well to 05 ng per well Both 96 and 384well assay formats yielded similar linearity and dynamic range indicating the suitability of the dualluciferase assay and SpectraMax i3x system for both formats Figure 4 Cellbased standard curves Cells transfected with both firefly and Renilla luciferases were seeded at densities from 195 to 25000 cells per well in a 96well plate and assayed using the DLR system Results were plotted as RLU vs number of transfected cells seeded per well Red plot firefly luciferase signal blue plot Renilla luciferase signal (r2 099 for both) Transfected cells seeded per well RLU Figure 5 Firefly luciferase signal in transfected cells was normalized to Renilla luciferase signal and the resulting normalized values graphed vs number of cells seeded per well Cells seeded per well Ratio of fireflyRenilla Cellbased assay Linearity of signal for both firefly and Renilla luciferases was observed across a broad range of cell densities from 195 to 25000 cells per well (Figure 4) The difference in magnitude of the luminescence signal between the two enzymes is due to the 101 ratio of fireflyRenilla vector used to transfect the cells as well as the different strengths of the SV40 (firefly vector) and TK (Renilla vector) promoters Figure 5 shows the firefly luciferase RLU values normalized to Renilla luciferase RLUs The normalized values were similar across the entire range of cell densities tested Contact Us Phone +18006355577 Web wwwmoleculardevicescom Email info@moldevcom Check our website for a current listing of worldwide distributors The trademarks used herein are the property of Molecular Devices LLC or their respective owners Specifications subject to change without notice Patents wwwmoleculardevicescomproductpatents FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES ©2015 Molecular Devices LLC 1015 Printed in USA Conclusion The results above demonstrate excellent sensitivity down to < 1 fg per well for firefly and Renilla luciferases and a 6decade dynamic range ensuring accurate measurement of luciferase signal over an extensive range of luciferase expression levels and cell densities The SpectraMax i3x reader features a cooled photomultiplier tube (PMT) for low background noise in luminescence When used in combination with the SpectraMax Injector Cartridge this system enables a wealth of flash type luminescence applications including reporter gene and other assays with exceptional sensitivity and dynamic range Analysis is done rapidly using a preconfigured SoftMax Pro protocol that displays each luciferase value and calculates normalized ratios for easy interpretation of results References 1 DeLuca MA and WD McElroy (1978) in Meth Enzymol 533 2 Matthews J C et al (1977) Purification and properties of Renilla reniformis luciferase Biochemistry 1658 3 httpswwwpromegacomproductspm dlreadyluminometersdlreadyvalidated luminometers《香当网》用户分享的内容，不代表《香当网》观点或立场，请自行判断内容的真实性和可靠性！该内容是文档的文本内容，更好的格式请下载文档