Buy Dual Luciferase Reporter Assay Kit, item number: G-MORV0010.100 from Assay Genie at Biomol! These viruses contain a firefly luciferase and eGFP cassette (Luc-P2A-eGFP) driven by a CMV promoter. Do notstore Luciferase Assay Reagent with dry ice. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. Note: Alternatively to the use of Luciferase Substrate from Promega, after cell lysis and transfer of the cell lysates into 96-Well microplate (see step 12i. The bioluminescence of this ATP-dependent luciferase is commonly used in reporter systems that are amenable to high-throughput screening. However, RLuc has a lower quantum yield compared with FLuc (6% versus 88%) and also shows a low enzymatic efficiency (9). Rapid Lysis of Cultured Mammalian Cells No need for scraping adherent cells or performing freeze-thaw cycles Minimal coelenterazine autoluminescence Lytic reagent of choice when processing cells for quantitation of firefly and Renilla luciferase activities using the Dual-Luciferase Reporter Assay System (Cat.# E1910) Size 30ml The sea pansy ( Renilla reniformis) is another luciferase (RLuc) that has been extensively investigated; RLuc uses coelenterazine as a substrate and does not require ATP to produce light. Add 9 l of Boost reagent from the RNA transfection kit to the RNA/OPTI-MEM mix, vortex briefly. using Fluc . At Gold Bio, we recommend a luciferase assay . Incubate at room temperature for about 30 min. Firefly (Fluc) and Renilla (Rluc) luciferases have accounted for the majority of such applications 7. Place 2 boxes of pipet tips, the plate of 3X firefly assay buffer, and a set of cell plates on the robot deck. Carefully transfer 20 l mix by pipetting into each well of cells cultured in a 24-well plate. Luciferase (firefly recombinant) is a light-generating enzyme that catalyzes an oxidative reaction, using luciferin, ATP, and molecular oxygen to produce oxyluciferin, which yields visible light. The Luminescence reads represent the Firefly luciferase activity. For plant tissue, quick-freeze in liquid nitrogen, grind the frozen tissue to a powder and resuspend at room temperature in 1X CCLR with further homo- genization.Remove the debris after cell lysis by a brief centrifugation.Assay the supernatant using standard assay conditions (Section IV). 2. The in vitro luciferin-luciferase assay is a relatively simple method that comprises cell transfection and expression of a luciferase probe targeted to a given organelle and the detection of the . Advantages Simplified Assay Optimization: Robust performance, improved storage and convenient size. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. If added later to the reaction mixture, CoA also promotes additional, secondary flashes (F raga, 2008). The Dual Luciferase Reporter Assay Kit sequentially measures the activity of Firefly and Renilla luciferases from the same sample. Place the plate in a plate reader and initiate reading. When injecting morpholinos, they should be prepared to a final concentration of 1-10 M. Copy the luminescence values into a spreadsheet program and plot the results. Includes: 10 vials Luciferase Assay Substrate (lyophilized) 10 10ml Luciferase Assay Buffer PRODUCT SIZE CAT.# Luciferase Assay System with Reporter Lysis Buffer 100 assays E4030 Each system contains sufficient reagents for 100 . Add 5l of 60mM EnduRen solution, place the tube back to 37 C. Luciferase Assay System 10-Pack 1,000 assays E1501 Contains sufficient reagents for 1,000 standard assays. Firefly Luciferase has an apparent molecular weight of 62 kDa, which is active as a monomer and does not require subsequent . Store at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. The Luciferase Assay Reagent generates light that is nearly constant for at least 1 minute and so the assay is compatible with measurement of luciferase using a single-tube luminometer or a multiwell plate luminometer with an auto-injector. Relative luciferase activity is the ratio between firefly luciferase and Renilla control luciferase. Place cells at RT for 10 min to complete lysis. The Luminescence reads represent the Renilla luciferase activity. Dissolve lyophilized luciferase by adding 10 ml of distilled water. Add 1.5 g of replicon RNA to OPTI-MEM in 15 ml tube, vortex briefly. Linear results are seen over at least eight orders of magnitude of enzyme concentration, and less than 10 -20 moles of luciferase can be measured under optimal conditions. Aliquot to avoid repeated freezing and thawing. Properties Database Information Handling & Safety Caution Bio-Glo Assay reagent is functionally tested for performance in the ADCC Reporter Bioassay and is intended for use with this or other bioassays. ), add 100 l of Firefly Luciferase Assay Buffer (Solution A, see materials and equipment) and 50 l Luciferin Substrate Solution (Solution B, see materials and equipment). or CPEB1. A luciferase release-based cytotoxicity assay was first described by Schafer et al. 1- Prepare the microinjection mix containing 20 ng/l of firefly luciferase and 2 ng/l Renilla reporter plasmids. Gently tap the plate on the side to mix wells and ensure that the bottom surface is covered with liquid. Aliquots are stable for 6 months after receipt at -20C. Dispense 50 l freshly prepared Stop & Glo Reagent (see Recipes) in the same plate, mix by pipetting 2 or 3 times and measure the Renilla luciferase activity by EnVision Multilabel Plate Reader. Add 50 L of the Luciferase Assay Reagent using a multichannel pipette to each well of the opaque plate. 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. of ten replicate samples and . Pre-warm 10 ml of DMEM with 10% FBS in a 15 ml tube in a water bath at 37 C. Store luciferase reagent at -20 C (see Note 3 ). The Luciferase Assay System is an extremely sensitive and rapid reagent for quantitation of firefly luciferase. Add 10 l master mix to DNA mix in each well of the 96-well plate and mix gently by pipetting. A luminometer is preferred, but not required as the assay is adaptable to scintillation counters. This kit is an Bio-Glo Luciferase Assay System Uses a Novel Firefly Luciferase Substrate, 5-fluoroluciferin. Firefly Luciferase Assay Kit * Luciferase Assay Kit (from the firefly Photinus pyralis) is an accurate, sensitive and easy method for studying gene reporter regulation and function in transformed cell lines in culture.*. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest. Incubate dissolved luciferase for 5 min at 4 C. Aspirate 40 l of 3X firefly assay buffer and add it to the first plate of cells, mixing thoroughly. Reconstituted Luciferase Assay Reagent (Luciferase Assay Substrate + Luciferase Assay Buffer) should be stored in aliquots at 20C for up to 1 month or at 70C for up to 1 year. The results are presented as the Renilla luciferase activity (A, B) or as the normalized luciferase activity (firefly/Renilla) (C). Avoid shaking. Thaw Luciferase Assay Reagent at temperatures below 25C and mix well before use. . Measure after . The Firefly Luciferase-eGFP Lentivirus are replication incompetent, HIV-based, VSV-G pseudotyped lentiviral particles that are ready to be transduced into almost all types mammalian cells, including primary and non-dividing cells. Each bar represents the mean S.E. Rna Immunoprecipitation Rip Lysis Buffer, supplied by Millipore, used in various techniques. Further dilutions can be made in assay buffer. June 1st, 2014 We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. But when Coenzyme A has been initially added to the assay buffer, it is able to prevent the fast reaction decay extending the half-life of the reaction from 2-5 minutes. The firefly luciferase reporter assay is initiated by adding an aliquot of lysate to Luciferase Assay Reagent II. Switching to new pipet tips, repeat for the second cell plate. h SIRT1-UTR luciferase constructs were co-transfected with shRNA negative control (Control) or shCPEB1-1/2 against CPEB1, and cells were . life around 0.5-1.0 min. Gentle swirl the bottle and mix for a homogeneous solution. Add Buffer Tango 10 to a final concentration of 0.5, and phenol red solution to a final concentration of 0.05%. Twenty-four hours after microinjection, the firefly and Renilla luciferase activity was measured using the Dual-Luciferase Reporter Assay System. For each strain or condition, use at least 100 animals to obtain more accurate results.