GC npo eia eeo TM GC npo eia eeo TM
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1. Note 1 Luc Buffers and Il are stable at 20 C for at least 6 months Freezing and thawing the reagents3 4 cycles won t affect the activity of the luciferases Aliquotting is recommended if more freeze thaw cycles are required Note 2 Working Solutions Buffers contain Substrates are stable at room temperature for 1 2 hours Prepare only the required amount of Working Solution before use Note 3 Light intensity is a measure of the rate of catalysis by the luciferases and is therefore temperature sensitive The temperature optimum for the activity of both luciferases is approximately room temperature 20 25 C so it is important that the reagents be equilibrated to room temperature before beginning measurements 1 Thaw Luc Buffer 5x and Luc Buffer Il 5x thoroughly at room temperature inverting the tube several times and then vortexing for 3 5 seconds Note Some pellets may appear in the Luc Buffer II 5x after thawing It is important to completely dissolve the pellets before using Incubation at 37 C for 5 10 minutes and more vortexing will be necessary to fully re dissolve the pellets 2 Dilute 1 5 in distilled water to make 1xLuc Buffer and 1xLuc Buffer Il Prepare 100 uL of each Buffer for each reaction well Duplicates or triplicates for each sample are recommended Example If you have 5 samples in duplicated reactions prepare 1 mL of 1xLuc Buffer and 1xLuc Buffer II by diluting 0 2mL of each 5xBuffers with 02. 100 reactions Shipping 300 reactions temperature 1000 reactions Lysis Buffer 10x kenak 20 C Cell Lysis buffer P Stable for at least 6 months 1 0 mLx2 Luc Buffer I 5x 1 0 mLx6 lee pack 20 C Firefly luciferase buffer hee Stable for at least 6 months mL x Storage temperature 100 uL Luc Sub l 100x 100 uLx3 Ice pack 20 C Firefly luciferase substrate Stable for at least 6 months 500 uLx2 1 0 mLx2 Luc Buffer ll 5x 1 0 mLx6 Ice pack 20 C Renilla luciferase buffer Stable for at least 6 months 10 mLx2 100 uL Luc Sub li 100x 100 uLx3 Ice pack 20 C Renilla luciferase substrate Stable for at least 6 months 500 uLx2 Luc Pair Duo Luciferase Assay Kit 2 0 User Manual lll Preparation of Cell Lysates Using Lysis Buffer The Lysis Buffer is supplied as a 10 X concentrate It may show turbid after thawing and mix which won t affect the luciferase assays Vortex 3 5 sec after thawing and prepare a sufficient quantity of the 1 X working concentration by adding 1 volume of 10 X Lysis Buffer to 9 volumes of distilled water and mix The diluted 1X Lysis Buffer may be stored at 20 C for 1 2 months however we recommend preparing the volume of Lysis Buffer required just before use A Lysis of Cells Cultured in Multi well Plates 1 Determine transfection parameters i e plated cell density and subsequent incubation time such that cells are 80 95 confluent at the desired time of lysate preparation Remove
3. 8 mL ddH2O respectively Preparing a little extra may be helpful to avoid buffer shortage caused by pipetting error 3 Prepare the FLuc and RLuc Assay Working Solution e g 10 samples by adding 10 uL of Luc Sub and II 100x to 1 mL of 1xLuc Buffer and 1xLuc Buffer Il respectively Mix well by inverting the tube several times 4 Incubate at room temperature for 5 minutes capped and protected from light before adding to the samples Note The RLuc Assay Working Solution will be used after reading the FLuc assay It can be kept at room temperature as long as 1 hour if properly capped and protected from light Luc Pair Duo Luciferase Assay Kit 2 0 User Manual V Assay Procedure 10 11 Set up the luminometer Follow the manual associated with your plate reader Set the measurement for 1 2 seconds of integration Pipette the cell lysis samples 20 uL per well into a 96 well white opaque or black plate or luminometer tubes Add the FLuc Assay Working Solution from step IV 4 100 uL per well or tube to the samples Gently pipette up and down mix the sample and assay solution Do not vortex Note If you have many samples and use 96 well plates we recommend using a multi channel pipette in order to reduce the time between additions of Assay Working Solution to each well Auto Injector is not recommended for this kit Incubate at room temperature for 3 5 minutes and proceed with the measurement Note Read the plate s
4. firefly luciferase activity will be at least 80 of its initial activity while Renilla luciferase activity will be at least 95 of its initial activity e Versatility The system has been designed for use with many different eukaryotic vertebrates lower invertebrates cell lines and is optimized for use on micro plate readers for high throughput screening assays Single tube samples can also be used e Low background The system produces very limited background luminescence No subtraction is required from readings e Simplicity Renilla luciferase buffer and substrate contains the quencher for firefly luciferase activity This allows for a quick two step assay Figure 2 e Reproducibility This system is designed to yield reliable linear results for a concentration range over several orders of magnitude Figure 3 Luc Pair Duo Luciferase Assay Kit 2 0 User Manual B A GeneCopoeia Luc Pair 2 0 aman Competitor Dual Luc System 1 000 000 000 000 m FUC 4 aaa REUC Kad ss 100 000 2 W F amp cK 10 000 eoep _ 1G O00 ee E A 0 0 20 30 40 5 69 8 19 20 30 40 50 50 Min Min Figure 1 Stability of firefly luciferase and Renilla luciferase signals using GeneCopoeia GCI Luc Pair Duo Luciferase Assay Kit 2 0 HEK 293 cells were transfected with GeneCopoeia pEZX MT06 miRNA reporter vector for 48 hours A The FLuc and RLuc activity was measured as described in the procedure B The Competitor s dual lu
5. the growth medium from the cultured cells and gently apply a sufficient volume of phosphate buffered saline PBS to wash the surface of the culture vessel Swirl the vessel briefly to remove detached cells and residual growth medium Completely remove the rinse solution before applying Lysis Buffer 2 Dispense into each culture well the minimum volume of 1 Xx Lysis Buffer required to completely cover the cell monolayer The recommended volumes of 1 X Lysis Buffer to add per well are as follows Culture Plate 1X Lysis Buffer uL Te n 500 12 well 250 5 0 Note The Lysis Buffer provided in the kit is sufficient for directly lysing cells in 24 48 or 96 well culture plates If a 6 well or 12 well plates are used we recommend either purchasing more Lysis Buffer Cat No LPFR LB100 or harvesting cells by scraping or trypsinization according to the procedures in III B below 3 Place the culture plates on a rocking platform or orbital shaker with gentle rocking shaking to ensure complete and even coverage of the cell monolayer with 1 lt Lysis Buffer Rock the culture plates at room temperature for 10 15 minutes Note1 If cell clumps appear pipetting several times could be helpful to disperse the cells Alternatively collect the cell lysates including cell clumps in tubes and vortex 5 10 sec after cooling down on ice Overgrown cells are more resistant to complete lysis and typically require an increased volume of lysis buffer to e
6. within 5 minutes after the incubation If using single luminometer tubes make sure the incubation and processing times before the luminescence detection are identical for all samples Save the reading if the luminometer reader does not automatically record the readings Remove the plates or luminometer tubes Add the RLuc Working Solution from Step IV 4 100 uL per well or tube to the plates or tubes from Step V 6 Gently pipette up and down or tap the plate tube several times to mix the sample and assay solution Do not vortex Note If you have many samples and use 96 well plates we recommend using a multi channel pipette in order to reduce the time between additions of Assay Working Solution to each well Auto Injector is not recommended for this kit Incubate at room temperature for 3 5 minutes and proceed with the measurement Note Read the plate s within 5 minutes after the incubation If using single luminometer tubes make sure the incubation and processing times before the luminescence detection are identical for all samples Record the reading if the luminometer reader does not automatically save the readings Remove the plates or luminometer tubes Calculate the ratio of luminescence from the firefly luciferase to the Renilla luciferase IMPORTANT NOTE Because the luminescent signals are affected by assay conditions raw results should be compared only between samples measured at the same time and using the same medium seru
7. GeneCopoeia Expressway to Discovery Luc Pair Duo Luciferase Assay Kit 2 0 For luciferase assays Cat No LPFR P010 100 reactions Cat No LPFR P030 300 reactions Cat No LPFR P100 1000 reactions User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2015 GeneCopoeia Inc Luc Pair Duo Luciferase Assay Kit 2 0 User Manual USER MANUAL Luc Pair Duo Luciferase Assay Kit 2 0 Introduction and Principles ll Contents and Storage lll Preparation of Cell Lysates Using Lysis Buffer IV Preparation of FLuc and RLuc Assay Working Solution V Procedure VI Limited Use License and Warranty I Introduction and Principles The study of transcriptional regulation using reporter gene expression is common and necessary in cell biology research and pharmaceutical discovery Luciferase is the most widely used genetic reporter for gene expression studies due to several advantages including 1 2 3 4 5 6 high sensitivity in a large dynamic range natural absence from mammalian cells consistent reproducibility cost effectiveness simple assay format suitable for high throughput screening Firefly and Renilla luciferases have been widely used as co reporters for normalization studies because both assays are quick easy and sensitive Firefly and Renilla luciferases are ideal co report
8. Longer stability of the enzyme substrate complex allows greater flexibility in monitoring true repressive events Further biological variation and stochastic events may add noise thereby reducing the differences in observed luciferase activity Thus normalizing the expression of an experimental reporter to the expression of an independent control reporter can help differentiate between true signal and nonspecific cellular responses Normalization is also needed for adjusting differences in transfection efficiencies and cell viability GeneCopoeia has leveraged the differences in firefly and Renilla enzyme structures and substrates to optimize and develop a convenient system for measuring two luciferase activities in succession The assay measures the activities of firefly and Renilla luciferases sequentially from a single sample The firefly luciferase luminescence is produced by one reagent while a second reagent simultaneously quenches the firefly luciferase and produces Renilla luciferase luminescence The GeneCopoeia Luc Pair Luciferase Kit 2 0 development team incorporated several features into the reagents to enhance product performance and convenience including the following e Enhanced stability The reagents have been developed so that the signals for firefly and Renilla luciferases exhibit greater stability than the original Luc Pair reagents and competitor kits Figure 1 30 minutes after addition of the appropriate reagents at 22 25 C
9. ciferase assay kit was used in comparison Firefly Luciferase activity before and after the addition of Rluc uter Before addition of Afer adicition cr Rluc SuPer Rieue buffer Figure 2 Firefly luciferase activity is quenched with the addition of Buffer Il HEK 293 cells were transfected with GeneCopoeia pEZX MT06 miRNA reporter vector for 48 hours The firefly luciferase activity was measured as described in the procedure Afterwards 1x Luc Buffer II without substrate was added to the wells followed by count reading in a luminometer Luc Pair Duo Luciferase Assay Kit 2 0 User Manual Side by Side Comparison of Genecopoeia s Luc Pair 2 0 vs the feading competitor A Fluc Assay B RLuc Assay 40 000 23 000 R 0 9987 R 0 9767 eee w 20 000 30 000 3 400 000 oe amp 2 R 0 9992 3 Ga R 0 9999 _ 390 000 5 20 000 7 5 V 10 000 206 000 amp E 8 10 000 5 000 190 900 0 o e 0 0 4 2 3 4 5 o 0 t 2 3 4 5 6 Celi number x 10 Celt Number x 107 Figure 3 Linear relationship between emitted light and amount of luciferase expression vector transfected in HEK293 HEK 293 cells were transfected with GeneCopoeia pEZX MT06 miRNA reporter vector for 48 hours The FLuc A and RLuc B activities were measured in lysates from different numbers of cells after incubation for 3 min as described in the procedure ll Contents and Storage Cat Nos LPFR P010 LPFR P030 and LPFR P100 Quantity Contents
10. ers because they have distinct evolutionary origins and very different enzyme structures and substrates This renders cross reactivity obsolete Firefly Photinus pyralis luciferase has been proven to be an ideal reporter for monitoring both promoter activity and post transcriptional regulation in the control of gene expression It is a cytoplasmic enzyme with a molecular weight of 61 kDa and catalyzes the following reaction HO aio 3 Na a Firef ty HO a a _ 5 Na fo LO fF Luciferase Y DON fy l p lt Re CO es pa AMP PP CO Light D Luciferin oxy Luciferin Luc Pair Duo Luciferase Assay Kit 2 0 User Manual The intensity of light emission is proportional to the amount of luciferase and is measured using a luminometer or multi function micro plate reader Renilla Renilla reniformis luciferase is a 36 kDa monomeric protein which requires no post translational processing Therefore it can function as a real time transcription reporter It catalyzes the following reaction 2 Ny ae ae CO Light Em HO A Ca Renilia O cr Luciferase Il i HO F N N i N Coelenterazine Coelenteramide Using this assay system allows one to monitor the transcriptional activation of cis elements in proximity to the gene of interest However it has been more difficult to measure the transcriptional repression via 3 UTR regulation of genes since the enzyme substrate activity window is relatively small
11. m combination Incorporation of consistent control wells on each plate provides the ability to calculate a normalized firefly luminescence Renilla luminescence ratio for each sample well These normalized ratios will remain essentially constant 410 for samples in a plate measured during the 1 hour measurement window Luc Pair Duo Luciferase Assay Kit 2 0 User Manual VII Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Luc Pair Duo Luciferase Assay Kit 2 0 the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial products or deliver information obtained in service without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is p
12. nsure complete lysis Note2 The firefly and Renilla luciferases contained in the cell lysates are stable for at least 30minutes at room temperature 22 C and up to 2 hours on ice 70 C is recommended for long term storage Subjecting cell lysates to more than 3 freeze thaw cycles may result in gradual loss of luciferase reporter enzyme activities 4 Transfer the lysate to a tube or vial for further handling and storage Alternatively reporter assays may be performed directly in the 96 well culture plate if the plates are compatible with the type of luminometer being used Luc Pair Duo Luciferase Assay Kit 2 0 User Manual B Lysis of Cells in tubes 5 For cells cultured in suspension or cells harvested by scraping or trypsinization Collect 1 2 10 cells in 1 5mL tubes rinse cells with 1mL of PBS buffer spin at 500g for 5minutes and completely remove the rinse solution 6 Add 50 100uL of 1 X Lysis Buffer to make 2 Xx 10 cells uL stand on ice for 2 5 min vortex 5 10 seconds to completely lyse the cells Note The firefly and Renilla luciferases contained in the cell lysates are stable for at least 30minutes at room temperature 22 C and up to 2 hours on ice 70 C is recommended for long term storage Subjecting cell lysates to more than 3 freeze thaw cycles may result in gradual loss of luciferase reporter enzyme activities 7 Proceed to Luciferase assay IV Preparation of FLuc and RLuc Assay Working Solution
13. roven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed to providing our customers with high quality products If you should have any questions orconcerns about any GeneCopoeia products please contact us at 301 762 0888 2015 GeneCopoeia Inc GeneCopoeia Products are for Research Use Only Copyright 2015 GeneCopoeia Inc Trademarks GeneCopoeia Luc Pair and GeneCopoeia Inc UMLPRFM002 021815
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