Rapid detection of firefly luciferase activity with amazing sensitivity and dynamic range.
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Rapid Detection of Firefly Luciferase Activity
NanoLuc® and NanoBiT® Luciferase Substrate for Animal Imaging Studies
PCR Master Mix(a,b) includes Nuclease-Free Water and PCR Master Mix, 2X. PCR Master Mix is a premixed, ready-to-use solution containing Taq DNA Polymerase, dNTPs, MgCl2 and reaction buffers at optimal concentrations for efficient amplification of DNA templates by PCR. The PCR Master Mix has been optimized for use in routine PCR for amplifying DNA templates in the range of 0.2–2kb.
The CellTiter-Glo® Luminescent Cell Viability Assay(a,b) is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells. The CellTiter-Glo® Assay is designed for use with multiwell formats, making it ideal for automated high-throughput screening (HTS), cell proliferation and cytotoxicity assays. The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing. The homogeneous "add-mix-measure" format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The CellTiter-Glo® Assay generates a "glow-type" luminescent signal, which has a half-life generally greater than five hours, depending on cell type and medium used. The extended half-life eliminates the need to use reagent injectors and provides flexibility for continuous or batch mode processing of multiple plates. The unique homogeneous format avoids errors that may be introduced by other ATP measurement methods that require multiple steps.
The Dual-Luciferase® Reporter (DLR™) Assay System(a–f) provides an efficient means of performing two reporter assays. In the DLR™ Assay, the activities of firefly (Photinus pyralis) and Renilla (Renilla reniformis or 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 luminescent signal lasting at least one minute. After quantifying the firefly luminescence, this reaction is quenched, and the Renilla luciferase reaction is initiated simultaneously by adding Stop & Glo® Reagent to the same sample. Both assays can be completed in about 4 seconds using a luminometer with reagent auto-injectors. In the DLR™ Assay System, both reporters yield linear assays with attomole (<10−18) sensitivities and no endogenous activity 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. The pGL4 and phRL series of synthetic Renilla Luciferase Reporter Vectors are designed for use with the DLR™ Assay Systems. A Renilla luciferase vector with constitutive expression may be used in combination with any experimental firefly luciferase vector to co-transfect mammalian cells.
High-throughput quantitation of firefly (Photinus pyralis) luciferase expression in mammalian cells is commonly performed by batch processing of 96- and 384-well plates. Steady-Glo® Luciferase Assay System(a,b,c) is designed for this purpose by providing long-lived luminescence when added to cultured cells. The homogeneous assay provides signal half-lives of over 5 hours in commonly used cell culture media without prior sample processing. Throughput rates of several thousand samples per hour may be achieved with high reproducibility under standard laboratory conditions.
Experience improved PCR performance with GoTaq® DNA Polymerase products. GoTaq® DNA Polymerase is a proprietary formulation of Taq DNA polymerase that gives robust amplification equal to and in some cases superior to that of standard Taq DNA polymerase. GoTaq® DNA Polymerase comes in a variety of formulations designed to give you maximum flexibility, control and convenience.
The CellTiter-Blue® Cell Viability Assay provides a homogeneous, fluorescent method for monitoring cell viability. The assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). Nonviable cells rapidly lose metabolic capacity and thus do not generate a fluorescent signal. The homogeneous assay procedure involves adding the single reagent directly to cells cultured in serum-supplemented medium. After an incubation step, data are recorded using either a plate-reading fluorometer (preferred) or spectrophotometer.
The PureYield™ Plasmid Midiprep System is designed to isolate transfection-quality plasmid DNA. The system provides a rapid method for purification of 100–200μg of plasmid DNA from 50ml bacteria culture. Plasmid DNA can be purified in as little as 30 minutes with the vacuum protocol, greatly reducing the time spent on purification compared to silica resin or other membrane-column methods. An alternative protocol allows purification of over 400μg of high-copy-number plasmid from 250ml of E. coli culture. The PureYield™ Plasmid Midiprep System incorporates a unique Endotoxin Removal Wash designed to remove substantial amounts of protein, RNA and endotoxin contaminants from purified plasmid DNA. Removal of contaminants improves the robustness of sensitive applications such as eukaryotic transfection, in vitro transcription and coupled in vitro transcription/translation (e.g., TnT® Quick Coupled Transcription/Translation System). Purification is achieved without isopropanol precipitation of purified plasmid DNA or extensive centrifugation, providing rapid purification from a single method. The system has been designed for use with centrifugation or vacuum (e.g., the Vac-Man® Laboratory Vacuum Manifold). PureYield™ Plasmid Midiprep System Features & Benefits: Improved Productivity - Vacuum protocol allows plasmid DNA purification in as little as 30 minutes. Confidence in Results - High purity and concentration of plasmid DNA gives proven performance in transfection, in vitro expression and other molecular biology applications. Ease of Use - Simple protocol eliminates tedious high-speed centrifugation, gravity-drip columns, and post-elution alcohol precipitation. Flexibility - PureYield™ membrane column allows purification of large amounts of plasmid DNA, exceeding the capabilities of other midiprep systems.
The CellTiter 96® AQueous One Solution Cell Proliferation Assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96® AQueous One Solution Reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(a)] and an electron coupling reagent (phenazine ethosulfate; PES). PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution. The CellTiter 96® AQueous Assay uses phenazine methosulfate (PMS) as the electron coupling reagent, and PMS Solution and MTS Solution are supplied separately. PES has enhanced chemical stability, which allows it to be combined with MTS to form a stable solution. Assays are performed by adding a small amount of the CellTiter 96® AQueous One Solution Reagent directly to culture wells, incubating for 1–4 hours and then recording absorbance at 490nm with a 96-well plate reader. The quantity of formazan product as measured by the amount of 490nm absorbance is directly proportional to the number of living cells in culture.
The Dual-Glo® Luciferase Assay System is a homogeneous reagent system that enables fast and simple quantitation of a stable luminescent signal from two reporter genes in a single sample. This convenient "add-and-read" system generates both firefly and Renilla luciferase luminescence signals from cells that have not been preconditioned or prelysed. With the Dual-Glo® System, internal controls can be established to minimize sample variability by reducing false positive and false negative readings caused by nonspecific factors such as cytotoxicity. In the Dual-Glo® Luciferase Assay, the activity of the primary reporter is correlated with the effect of specific stimuli, and the activity of the co-transfected control reporter provides an internal control to normalize results.
Use with ADP-Glo™ Assay for bioluminescent detection of kinase activity
ProteaseMax™ Surfactant is a high-quality reagent for use in protein and peptide sample preparation. ProteaseMax™ Surfactant solubilizes proteins and ensures fast and improved protein digestion with trypsin or chymotrypsin. It is designed to degrade over the course of a digestion reaction, yielding products that are compatible with downstream methods such as mass spectrometry (MS) and liquid chromatography (LC). For in-gel protein digestion, ProteaseMax™ Surfactant also improves the recovery of peptides. ProteaseMax™ Surfactant can be used with existing in-gel or in-solution digestion protocols.
The pGEM®-T Easy Vector Systems are convienent systems to clone PCR products. They offer all of the advantages of the pGEM®-T Vector Systems with added convieneice of recognition sites for EcoRI and NotI flankin the insertion site. Thus, several options exist to remove the desired insert DNA with a single restriction digestion. The pGEM(R)-T Easy Vectr System II contains JM109 Competent Cells in addition to all pf the pGEM(R)-T Easy Vector System I components. pGEM®-T Easy Vector Systems Features & Benefits: Flexibility - The multiple cloning site is flanked by restriction enzyme sites for BstZI, NotI and EcoRI, allowing three options to remove the insert with a single digest. Rapid Ligation - The 2X Rapid Ligation Buffer provided allows reactions to be completed in 1 hour at room temperature. Blue/White Screening - T7 and SP6 RNA polymerase promoters flank a multiple cloning region within the α-peptide coding region for β-galactosidase. Insertional inactivation of the α-peptide allows recombinant clones to be directly identified by color screening on indicator plates. f1 Origin of Replication - Allows preparation of single-stranded DNA.
The Promega CytoTox 96® Non-Radioactive Cytotoxicity Assay is a colorimetric alternative to radioactive cytotoxicity assays. The CytoTox 96® Assay quantitatively measures lactate dehydrogenase (LDH), a stable cytosolic enzyme that is released upon cell lysis, in much the same way as [51Cr] is released in radioactive assays. Released LDH in culture supernatants is measured with a 30-minute coupled enzymatic assay that results in the conversion of a tetrazolium salt (INT) into a red formazan product. The amount of color formed is proportional to the number of lysed cells. Visible wavelength absorbance data are collected using a standard 96-well plate reader. The assay can be used to measure membrane integrity for cell-mediated cytotoxicity assays in which a target cell is lysed by an effector cell, or to measure lysis of target cells by bacteria, viruses, proteins, chemicals, etc. Features of the Promega CytoTox 96® Non-Radioactive Cytotoxicity Assay: • Non-Radioactive: Requires no radioactive waste disposal or [51Cr]. • Save Time: Eliminates labeling of target cells prior to experiment. • Use Standard Equipment: Collect absorbance (visible wavelength) data with a standard 96-well plate reader. • Adapt to Your Needs: Used for a variety of applications including measurement of: 1) cell-mediated cytotoxicity; 2) chemical-mediated cytotoxicity; and 3) total cell number. • Gain Sensitivity: Can reveal early, low-level damage to cell membranes that is often missed with other methodologies. Applications of the CytoTox 96® Non-Radioactive Cytotoxicity Assay: • Cytotoxicity. • Chemosensitivity. • Total cell number determination.
