Luciferase Reporter Assay Kit
Contents
1. an assay dilute an aliquot of 3X Cell Lysis Buffer to 1X with deionized or distilled water see Section V A Reagents provided are sufficient for 100 assays e 10 ml Substrate A e 10 ml Substrate B e 50 ml 3X Cell Lysis Buffer lll Additional Materials Required The following materials are required but not supplied Phosphate buffered saline PBS pH 7 4 Final conc To prepare 2 L of solution Na HPO 58 mM 16 5 g NaH PO 17 mM 41 g NaCl 68 mM 8 0 g Dissolve the above components in 1 8 L of deionized H O Adjust to pH 7 4 with 0 1 N NaOH Add deionized H O to final volume of 2 L Store at room temperature e Centrifuge for collecting cells 1 5 ml microcentrifuge tubes 0 5 ml microcentrifuge tubes or 96 well flat bottom microtiter plate Chemiluminescence assays are generally performed in 0 5 ml microcentrifuge tubes Alternatively reactions can be performed in white opaque 96 well flat bottom microtiter plates such as those from Xenopore or Costar e Luminometer tube or plate scintillation counter or x ray film e Lysozyme for bacterial cell lysis Protocol PT3392 1 www bdbiosciences com BD Biosciences Clontech Version PR2Y278 5 Luciferase Reporter Assay Kit User Manual IV General Considerations Ensure that all reagents have reached room temperature before performing assays e Donotrepeatedly freeze thaw sample extracts Loss of luciferase activity will result e We strongly2. ml 1X Cell Lysis Buffer and vortex to resuspend cell pellet Let stand at room temperature for 5 10 min then centrifuge lysate at 14 000 rpm at room temperature for 1 min to remove insoluble debris As with eukaryotic samples assays should be performed within 20 min For measurements that require longer time points or for assays that are to be completed at a later date extracts may be stored for up to one month at 70 C C Luciferase Assay 1 2 Place 20 100 ul cell extract into an assay cuvette Be sure to use the same volume for each sample If measurement will be performed on a luminometer or scintillation counter the recommended measurement time is 10 30 sec photo graphic or CCD type instruments typically require exposures as long as 5 min Follow the step below that is appropriate for your instrument a lf your luminometer contains a single automatic injector manually add 100 ul of Substrate A to the assay cuvette and automatically inject 100 ul of Substrate B within 10 min Set the delay after the injection and before measurement to 1 or 2 sec b If your luminometer contains two automatic injectors inject 100 ul of Substrate A first followed by 100 ul of Substrate B Set the delay between injections to 1 or 2 sec Set the delay after the second injection and before measurement to 1 or 2 sec c If no automatic injectors are used manually add 100 ul of Substrate Ato the assay cuvette Immediately befo
3. or animals By providing faster results lower costs and over a 1 000 fold increase in sensitivity the luciferase assay has largely replaced the standard C chloram phenicol acetyltransferase CAT assay Our Luciferase Reporter Assay Kit provides a simple means for detecting luciferase activity in transformed bacteria or transfected eukaryotic cells The kit includes a firefly luciferase substrate formulation and an optimized cell lysis buffer This buffer enhances luciferase recovery and activation when used with in vitro assays The unique formulation of this kit provides high sensitivity constant light output as well as convenience and consistency when working with multiple samples Background Firefly luciferase has been reliably expressed as a reporter gene from many expression vectors and in a variety of organisms Its major use has been to characterize gene regulation primarily transcriptional control by correlating variations in luciferase activity with the regulation of promoter and enhancer elements Performed under optimal conditions the peak height and integrated total light output from a reaction is proportional to the amount of functional luciferase enzyme This results in a direct relationship between the amount of light emitted from the sample and the transcriptional activity of the regulatory elements Firefly luciferase catalyzes the oxidative carboxylation of luciferin a reaction with the highest efficiency of any
4. Luciferase Reporter Assay Kit User Manual Cat No K2039 1 PT3392 1 PR2Y278 Published 11 22 2002 Luciferase Reporter Assay Kit User Manual Table of Contents l Introduction ll List of Components lll Additional Materials Required IV General Considerations V Assay Procedure A Eukaryotic Cell Lysis B Bacterial Cell Lysis C Luciferase Assay VI Troubleshooting Guide VII References Vill Related Products 00000 Y NO OF 0 _ mb Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use BD Biosciences Clontech products may not be resold modified for resale or used to manufacture commercial products without written approval of BD Biosciences Clontech BD Adeno X BD Creator BD Living Colors BD Mercury BD RevTet OffTM BD RevTet On BD Tet Off and BD Tet On are trademarks of Becton Dickinson and Company O 2002 BD BD Biosciences Clontech www bdbiosciences com Protocol PT3392 1 2 Version PR2Y278 Luciferase Reporter Assay Kit User Manual I Introduction Firefly beetle Photinus pyralis luciferase is one of the most popular reporter molecules used in molecular biology and biochemistry Gould amp Subramani 1988 Vieites et al 1994 Gailey et al 1997 Luciferase can be used to monitor promoter response activity in bacteria cultured cells and transgenic plants
5. ctivity Lemasters amp Hackenbrock 1977 To over come this rapid extinction the Luciferase Reporter Assay Kit includes Coenzyme A CoA which displaces the inhibiting oxyluciferin product substrate from the enzyme facilitating its turnover Airth et a 1958 Inclusion of CoA in the luciferase assay yields anearly constant light emission rather than the typical flash kinetics Figure 1 resulting in a more sensitive assay By providing CoA along with ATP Mg and buffer in a preformulated substrate mix the Luciferase Reporter Assay Kit ensures maximal sensitivity along with an increased ease of handling The Luciferase Reporter Assay Kitis suitable for use with any standard transfection experiment utilizing firefly luciferase as a reporter 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 Relative Light Units RLU 1 10 20 30 40 50 60 Time Seconds Figure 1 Effect of CoA on firefly luciferase kinetics Basic luciferase assay A and luciferase assay modified by the addition of CoA B BD Biosciences Clontech www bdbiosciences com Protocol PT3392 1 4 Version PR2Y278 Luciferase Reporter Assay Kit User Manual ll List of Components Prior to reconstitution store all reagents at 20 C Substrates A and B must each be reconstituted in 10 ml deionized or distilled water After reconstitution Substrates A and B may be stored as aliqouts either at 20 C for 6 months or at 4 C for 5 days Before starting
6. hat a purified luciferase standard does not necessarily represent the exact amount of luciferase produced by transfected cells since the specific activity of the expressed luciferase may differ from the purified luciferase BD Biosciences Clontech www bdbiosciences com Protocol PT3392 1 6 Version PR2Y278 Luciferase Reporter Assay Kit User Manual V Assay Procedure PLEASE READ ENTIRE PROTOCOL BEFORE STARTING Important Equilibrate all reagents to room temperature before starting the assay A Eukaryotic Cell Lysis This protocol is optimized for use with eukaryotic cell cultures For a lysis protocol for bacterial cells please see Part B below 1 Prepare an adequate amount of 1X Cell Lysis Buffer by diluting 1 part 3X Cell Lysis Buffer into 2 parts distilled or deionized water see Table I The following protocol is designed for use with adherent cultures growing in 35 mm tissue culture plates If you are using plates wells or flasks of a different size adjust the volume proportionally Remove media from cell culture plates and rinse twice with phosphate buffered saline PBS without Ca and Mg see Section III for recipe Add 200 ul 1X Cell Lysis Buffer to cells and shake at room temperature for 15 20 min Alternatively cells may be lysed at 4 C to minimize protease activity If performing lysis at 4 C allow cell lysate to reach room temperature before continuing with protocol Dislodge cells by
7. in Saccharomyces cerevisiae Yeast 10 1321 1327 BD Biosciences Clontech www bdbiosciences com Protocol PT3392 1 10 Version PR2Y278 Luciferase Reporter Assay Kit User Manual Vill Related Products For the latest and most complete listing of all BD Biosciences Clontech products please visit www bdbiosciences com BD Mercury Reporter Systems e BD Mercury Pathway Profiling Luciferase System 1 K2049 1 e BD Mercury Pathway Profiling Luciferase System 2 K2052 1 e BD Mercury Pathway Profiling Luciferase System 3 K2053 1 e BD Mercury Pathway Profiling Luciferase System 4 K2056 1 BD Mercury Pathway Profiling Luciferase System 5 K2057 1 Tet Expression Systems e BD Tet Off Gene Expression System K1620 1 BD Tet On Gene Expression System K1621 1 pBI L Bidirectional Tet Vector 6151 1 Retroviral Expression Systems BD pRevtTet Off System K1626 1 BD pRevTet On System K1627 1 BD Adeno X Tet Off Expression System K1651 1 e BD Adeno X Tet On Expression System K1652 1 BD Creator DNA Cloning Kits e BD Creator pDNR 1 Cloning Kit K1670 1 BD Living Colors Vectors e pEGFPLuc Vector 6169 1 Protocol PT3392 1 www bdbiosciences com BDBiosciencesClontech Version PR2Y278
8. known bioluminescence reaction Seliger amp McElroy 1960 Atthe optimal reaction pH of 7 8 light emission peaks at 562 nm This form of light emission yields a very sensitive non radioactive assay The Assay Firefly luciferase is a 62 000 dalton protein which is active as a monomer and does not require subsequent processing for its activity However several factors may affect the sensitivity and success of the assay including pH temperature and substrate concentration To ensure maximum sensitivity the assay is performed in the presence of excess ATP luciferin and Mg in a buffer that will maintain a pH of 7 8 For measurement of expressed luciferase activity in vitro luciferase is extracted from transfected cells through cell lysis A typical firefly luciferase assay is then carried out in an assay cuvette ATP Mg and buffer are added to the lysate either separately or as a preformulated solution The luminescent reaction is then triggered by an injection of luciferin and the emitted light is recorded Protocol PT3392 1 www bdbiosciences com BD Biosciences Clontech Version PR2Y278 3 Luciferase Reporter Assay Kit User Manual I Introduction continued When luciferin is added to a sample containing luciferase there is an immediate light flash that reaches peak intensity at 0 3 0 5 seconds and then decays rapidly This rapid exponential decay is caused by the reaction product oxyluciferin which inhibits luciferase a
9. re measurement manually add 100 ml of Substrate B to the assay cuvette The time between adding Substrate B and start of measurement should be as short as possible and consistent from sample to sample BD Biosciences Clontech www bdbiosciences com Protocol PT3392 1 8 Version PR2Y278 Luciferase Reporter Assay Kit User Manual VI Troubleshooting Guide A Intra assay Variability Pipetting error Temperature changes Allowing sample and buffer to sit for extended periods of time Reagent degradation Use larger sample volumes to minimize variability caused by pipetting error Be sure all reagents have reached room temperature before performing assay Work quickly to minimize the time between adding and initiating the reaction Store all reagents at 20 C B Abnormally Low Light from Assay Improper pH Improper substrate concentrations Reagent degradation Presence of interfering substances C High Background Contaminated reagents Contaminated injector lines Protocol PT3392 1 Version PR2Y278 Test pH of each reagent and adjust to 7 8 if necessary Check that the correct volume of each reagent is being added to the assay reaction and adjust if necessary Store all reagents at 20 C Be sure to wash cells thoroughly with PBS 2 3 times before performing lysis Reagents may become contaminated by carry over from pipette tips Be sure to change tips between reaction component
10. recommend using the Cell Lysis Buffer and protocols supplied with this kit Sonication or other methods of cell lysis may reduce the sensitivity of the assay e Optimization of the Luciferase Assay Kit may be necessary for use with your equipment or samples Signal detection may become saturated when mea suring very high light emitting samples in a luminometer or scintillation counter If this occurs dilute your sample with 1X Lysis Buffer and repeat the asSay e Chemiluminescent detection of luciferase activity can be performed with a luminometer tube or plate or a liquid scintillation counter LSC Use of an LSC may result in lowered sensitivity and increased variability between samples due to the need for manual addition of Substrate B Nguyen et al 1988 In addition it is necessary to make specific adjustments to the LSC for the correct detection of the luciferase signal Fulton amp Van Ness 1993 e Itis also possible to measure luciferase expression via exposure of x ray film to reactions performed in a white opaque 96 well flat bottom microtiter plate Xenopore or Costar e Measured levels of luciferase activity are normally stated in relative light units RLUs which do not represent an absolute value If you wish to correlate your relative experimental luciferase activities with an absolute value you must generate a standard curve for your measuring equipment using purified luciferase However it is important to be aware t
11. s and or samples Replace com ponent if necessary Flush injector lines thoroughly with distilled water www bdbiosciences com BD Biosciences Clontech 9 Luciferase Reporter Assay Kit User Manual VII References Airth R L Rhodes W C 8 McElroy W D 1958 The function of coenzyme A in luminescence Biochem et Biophys Acta 27 519 532 Gailey P C Miller E J amp Griffin G D 1997 Low cost system for real time monitoring of luciferase gene expression BioTechniques 22 528 534 Gould S J amp Subramani S 1988 Firefly luciferase as a tool in molecular and cell biology Analyt Biochem 175 5 13 Fulton R amp Van Ness B 1993 Luminescent reporter gene assays for luciferase and B galactosidase using a liquid scintillation counter BioTechniques 14 762 763 Lemasters J J amp Hackenbrock C R 1977 Kinetics of product inhibition during firefly luciferase luminescence Biochemistry 16 3 445 447 Nguyen V T Morange M 8 Bensaude O 1988 Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells Analyt Biochem 171 404 408 Seliger H H amp McElroy W D 1960 Spectral emission and quantum yield of firefly biolumines cence Arch Biochem Biophys 88 136 141 Vieites J M Navarro Garcia F P rez Diaz R Pla J amp Nombela C 1994 Expression and in vivo determination of firefly luciferase as gene reporter
12. scraping or pipetting and transfer to a 1 5 ml microcentrifuge tube Spin cells at 14 000 rpm at room temperature for 1 min to remove cellular debris Samples should be assayed within 20 min For measurements that require longer time points or for assays that are to be completed at a later date extracts may be stored for up to one month at 70 C TABLE I CULTURE PLATE CONVERSION Plate or Growth Relative Recommended Volume Flask Size Area cm2 Area 1X Cell Lysis Buffer 96 well 0 32 0 04 X 20 ul 24 well 1 88 0 25 X 50 ul 12 well 3 83 0 50 X 100 ul 6 well 9 4 1 20 X 200 ul 35 mm 8 0 1 00 X 200 ul 60 mm 21 2 60 X 500 ul 10 cm 55 7 00 X 1 0 ml T25 25 3 00 X 500 ul T75 75 9 00 X 1 2 ml Relative area is expressed as a factor of the growth area of a 35 mm culture plate Protocol PT3392 1 Version PR2Y278 www bdbiosciences com BD Biosciences Clontech 1 Luciferase Reporter Assay Kit User Manual V Assay Procedure continued B Bacterial Cell Lysis 1 Prepare 1 ml of 1X Cell Lysis Buffer for each 1 10 ml aliquot of bacterial culture by making a 1 3 dilution of 1 part 3X Cell Lysis Buffer to 2 parts distilled or deionized water Add lysozyme to a final concentration of 1 mg ml Centrifuge a 1 10 ml aliquot of bacterial culture If required an optimal volume may be determined after initial measurement of activity Remove and discard supernatant without disturbing cell pellet Add 1
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