pGL3 Luciferase Reporter Vectors Technical Manual #TM033

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V. Transfection of Mammalian CellsTransfection of DNA into eukaryotic cells may be mediated by cationic lipidcompounds (2), calcium phosphate (3,4), DEAE-dextran (3,5), or electroporation(4). Transfection systems based on cationic lipids (TransFast TransfectionReagent, Transfectam ® Reagent and Tfx Reagents) and calcium phosphate(Profection ® Mammalian Transfection System) are available from Promega. Formore information on these transfection reagents, please request the TransFastTransfection Reagent Technical Bulletin (#TB260), the Transfectam ® Reagent TechnicalBulletin (#TB116), the Tfx-Reagents Technical Bulletin (#TB216) or the ProFection ®Mammalian Transfection System Technical Manual (#TM012). All of thesedocuments are available on our web site at: www.promega.com/tbs/VI.Assay of Luciferase ActivityExperimental strategies using firefly luciferase may involve the analysis of afew samples per day or as many as several thousand samples per hour, andequipment used to measure luminescence may vary from inexpensive, singlesampleluminometers to high-end CCD luminometers. To support this widerange of applications, we have developed three luciferase assays with different,but complementary, characteristics: Luciferase Assay System (Cat.# E1500),Bright-Glo Luciferase Assay System (Cat.# E2610), Steady-Glo ® LuciferaseAssay System (Cat.# E2510), and ONE-Glo Luciferase Assay System (Cat.#E6110). Reagent choice depends on the relative importance of experimentalformat, assay sensitivity, and luminescence duration.Table 1. Characteristics of Promega Luciferase Assay Reagents.LuciferaseBright-Glo Steady-Glo ® Assay ONE-GloReagent Reagent Reagent ReagentFormat NH or H NH or H NH NH or HProcess continuous batch bench scale batch orcontinuousNumber of Steps 1 1 4 1Sensitivity highest lower higher highSignal Half-Life ~30 minutes ~5 hours ~12 minutes ~50 minutesPrecision High High High HighestCell Lysis Time ~2 minutes ~5 minutes NA ~3 minutesmaximum maximumNH = nonhomogeneous (first create a lysate); H = homogeneous; NA = not applicablePromega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USAToll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.comPrinted in USA.Part# TM033Revised 9/07 Page 9
VI.Assay of Luciferase Activity (continued)The Luciferase Assay System has long been the standard reagent for routinelaboratory analysis. Before using this reagent, cells from which the luciferase isto be measured must be washed and lysed. This reagent was optimized forhigh sensitivity in nonhomogeneous, single-sample measurements. TheLuciferase Assay System requires a luminometer fitted with injectors toefficiently measure luminescence in 96-well plates.The Bright-Glo, Steady-Glo ® and ONE-Glo Reagents were developed toperform assay reactions within multiwell plates and in the presence ofcomplete cell culture medium: no cell preparation steps such as washing orlysing are required before the luminescence reaction is initiated. All of these aresingle-step reagents, requiring only addition of the reagent before measuringluminescence. This makes them ideal reagents for efficient and precisequantitation in 96-, 384- and 1536-well plates.The Bright-Glo and Steady-Glo ® Reagents are complementary in theircharacteristics based on the inverse relationship between luminescenceduration and assay sensitivity (6). Generally, as the half-life of the luminescenceincreases, assay sensitivity decreases. The Steady-Glo ® Reagent provides longluminescence duration (changing only about 10% per hour); however, toachieve this long luminescence duration, the assay sensitivity must be reduced.This reagent was designed for experiments in which many microplates areprocessed as a batch.In contrast, the Bright-Glo Reagent provides high assay sensitivity withshorter luminescence duration (
Page 1 and 2: Technical ManualpGL3 Luciferase Rep
Page 3 and 4: I. DescriptionThe pGL3 Luciferase R
Page 5 and 6: III.B. pGL3-Enhancer VectorThe pGL3
Page 7 and 8: III.D. pGL3-Control VectorThe pGL3-
Page 9: IV.B. Preparation of pGL3 Vectors a
Page 13 and 14: VIII. AppendixVIII.A. Common Struct
Page 15: VIII.C. The pGL3 Vectors luc+ GeneM
Page 18 and 19: VIII.F. References1. Sambrook, J. e
Page 21: VIII.G. pGL3-Basic Vector Restricti
Page 25 and 26: VIII.I. pGL3-Promoter Vector Restri
Page 27: VIII.J. pGL3-Control Vector Restric
Page 30: pGL4 Luciferase VectorsProduct Size

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