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Secrete-PairTM Gaussia Luciferase Assay Kit

1. T T T T aj 0 5 10 15 20 25 30 35 Time Min Figure 3 GLuc and SEAP assays Cell culture medium was collected from cells transfected with wild type wt GLuc SEAP dual reporter clone 10 ul of the medium was used in each assay At the beginning the GLuc activity in Buffer GL H is about 4 6 times higher than that in Buffer GL S Then it quickly decays The GLuc activity in Buffer GL S however is much more stable Sample preparation 1 Transfect cells with single or dual reporter constructs using Endofectin GeneCopoeia Cat EFP1003 or other transfection reagents Duplicated transfections are recommended Note We suggest using 6 well or 12 well cell culture plates for transfection Other type of cell culture plates or dishes can also be used If co transfection is required the optimal working condition should be determined by the user The general recommendation is using reporter construct e g GLuc reporter clone and normalization control construct e g SEAP plasmid at 1 5 1 ratio 2 Change to fresh medium 24 hours after transfection After changing the medium you may start to treat or challenge the cells if specific conditions are to be tested 3 After a proper period of time 48 72 hours after transfection gently collect the cell culture medium for GLuc and SEAP luminescent assays Store the collected medium in 20 C if not use immediately The enzymes are stable at 20 C for at least one month 4 Optional 10 ul
2. User Manual IV Preparation Note 1 The 10x buffers must be thawed thoroughly at room temperature and vortex for 3 5 Sec The buffers may turn a little turbid which will not affect the assay before diluted to 1x working buffer The 1x working buffer without the substrate can be stored at 4 C for one week 2 The luminescence catalyzed by the luciferases or SEAP is temperature sensitive The optimal temperature for the activities of GLuc and SEAP is room temperature 20 25 C It is important that the 1x working buffer be equilibrated to room temperature before adding samples for luminescence detection 3 For the GLuc assay use either Buffer GL S for more stable activity or Buffer GL H for higher sensitivity The initial GLuc activity in Buffer GL H is about 4 6 times higher than that in Buffer GL S Fig 3 However the GLuc activity in Buffer GL S is much more stable It retains more than 90 of signal within the first 10 min while the activity in Buffer GL H dropped to less than 40 within the same period of time Fig 3 amp 4 Therefore we recommend starting the GLuc assay from Buffer GL S first especially for high throughput screen If the GLuc activity is too low you may consider using the Buffer GL H 4 For the SEAP assays the Buffer AP should be used GLuc and SEAP assays 70 000 60 000 4 SEAP 50 000 S a GL H 2 40 000 3 z 30 000 sa 20 000 e 10 000 e e 0 T T T
3. customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2014 GeneCopoeia Inc GeneCopoeia Products are for Research Use Only Copyright 2013 GeneCopoeia Inc Trademarks GeneCopoeia Secrete Pair GLuc ON miTarget GeneCopoeia Inc SPDA 140408
4. 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 proven 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
5. luminometer should be used The GL H buffer can be used to measure the activity of mGLuc for enhanced sensitivity The measurement should be completed within 1 2 min after adding the Assay Working Solution to the samples 1 Collect 0 1 0 2 ml of medium from each cultured cells in 1 5 ml tubes and place at room temperature 2 Thaw Buffer GL H 10x thoroughly at room temperature inverting the tube several times and then vortex for 3 5 Sec Dilute 1 10 in ddH20 to make 1x Buffer GL H Prepare 100 ul of 1x Buffer GL H for each reaction well Duplicates or triplicates for each sample are recommended For example If you have 5 samples in duplicated reactions preparing 1 ml of 1x Buffer GL H by diluting 0 1ml of 10x Buffer GL H with 0 9 ml ddH2O Preparing a little bit extra may be helpful to avoid buffer shortage caused by the pipetting error 3 Prepare the GLuc Assay Working Solution e g 10 samples by adding 10 ul of Substrate GL to 1 ml of 1x Buffer GL H Mix well by inverting the tube several times 4 Incubate at room temperature for 25 minutes capped and protect from light before adding to the samples 5 Setup the luminometer Set the measurement for 1 2 seconds of integration 6 Pipet culture medium samples 10 ul per well in duplicates or triplicates into a 96 well white opaque or black plate or luminometer tubes 7 Add the GLuc Assay Working Solution from Step 4 100 ul per well or tube to the samples from Step 6 Gen
6. of EF1A PG04 media included can be used as a positive control to test the illuminometer and the Secrete Pair dual luminescence assay kit Secrete Pair Luminescence Assay Kits User Manual V Gaussia Luciferase Assay Procedure Two GLuc assay buffers are provided in the kit to meet your specific research needs GLuc buffer selection guide GLuc assay buffer Purpose wtGLuc mGLuc Application For more stable Regular HTS manual GL S buffer activity Required Recommended assay Not recommended Regular HTS manual GL H buffer For higher unless activity is anen gner assay when handled sensitivity sensitivity is needed very low properly wtGLuc humanized wild type Gaussia luciferase mGLuc modified Gaussia luciferase It generates much more stable luminescence signal than the wtGLuc Fig 4 GeneCopoeia GLuc ON promoter clones and miTarget miRNA 3 UTR target clones uses mGLuc ome GL S wtGLuc GL S mGLuc meee w GL H a m GLH 120 L 100 ae g 80 4 salan 3 60 acc pa 404 ais 20 20 0 p Se ene we 0 T T T 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 Time Min Time Min Figure 4 Signal stabilities of wtGLuc and mGLuc assays using GL S or GL H buffers Cell culture medium was collected from cells transfected with either humanized wild type GLuc wtGLuc or modified GLuc mGLuc reporter clones 10 ul of the medium was used in each assay A
7. Expressway to Discovery Secrete Pair Dual Luminescence Assay Kit For parallel bioluminescence assays of Gaussia luciferase GLuc and secreted Alkaline Phosphatase SEAP Cat No SPDA D010 100 reactions Cat No SPDA D030 300 reactions Cat No SPDA D100 1000 reactions Secrete Pair Gaussia Luciferase Assay Kit For stable and sensitive assay of Gaussia luciferase activity Cat No SPGA G010 100 reactions Cat No SPGA G100 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 2014 GeneCopoeia Inc Secrete Pair Luminescence Assay Kits User Manual USER MANUAL Secrete Pair Dual Luminescence Assay Kit Secrete Pair Gaussia Luciferase Assay Kit Introduction and Principle Il Contents and Storage Ill Protocol Overview IV Preparation V GLuc Assay Procedure VI SEAP Assay Procedure VII Signal Normalization VIII Important Note IX References X Limited Use License and Warranty I Introduction and Principle Secrete Pair Dual Luminescence Assay Kit The Dual Luminescence Assay kit is designed to analyze the activities of Gaussia Luciferase GLuc and Secreted Alkaline Phosphatase SEAP using luminescent assays side by side from a single sample such as cell culture medium Both GLuc and SEAP are secreted reporter proteins Samples can be easily obtained
8. Luc signal stability comparison 100 4 S GL S Secrete Pair al as TR EE oie gt eM ms ompetitor 3 604 5 eesse Competitor H 2 7 N gt 2 gt 404 e z S cy T 20 ann w EE e 0 T RRR ESLLISE EINE Yee hl ae Tee 0 5 10 15 20 25 30 35 40 Time Min Figure 2 Comparison of GLuc signal stability in different buffer systems from Secrete Pair and a competitor Gaussia luciferase assay kit Cell culture medium was collected from cells transfected with the humanized wild type GLuc reporter clones 10 ul of the medium was used in each assay Two buffer systems of each kit were tested and the assays were performed according to the manufacturer protocols The percentage of signal retained Y axis is used as an indicator for signal stability For both kits the GLuc activities in buffers with a stabilizer S are much more stable than those in buffers without a stabilizer H However when compared side by side Secrete Pair buffer systems provide more stable GLuc signal than the competitor kit More than 90 of signal was retained within the first 10 minutes using GL S buffer from Secrete Pair blue and only about 70 of signal was retained using the competitor stable buffer green Secrete Pair Luminescence Assay Kits User Manual ll Contents and Storage Quantity 100 reactions Shipping Storage Dare Coments 300 reactions temperature temperature Secrete Pa
9. Protocol for enhanced signal stability using GL S buffer Note The GLuc activity is very stable in Buffer GL S for both wtGLuc and mGLuc Fig 4 It provides steady kinetics over a longer time period which is suitable for high throughput analysis as well as manually delivered assays 1 Collect 0 1 0 2 ml of medium from each cultured cells in 1 5 ml tubes and place at room temperature 2 Thaw Buffer GL S 10x thoroughly at room temperature inverting the tube several times and then vortex for 3 5 Sec Dilute 1 10 in distilled water to make 1x Buffer GL S Prepare 100ul of 1xBuffer GL S for each reaction well Duplicates or triplicates for each sample are recommended For example If you have 5 samples in duplicated reactions preparing 1 ml of 1x Buffer GL S by diluting 0 1ml of 10x Buffer GL S with 0 9 ml ddH2O Preparing a little bit extra may be helpful to avoid buffer shortage caused by the pipetting error 3 Prepare the GLuc Assay Working Solution e g 10 samples by adding 10 ul of Substrate GL to 1 ml of 1xBuffer GL S Mix well by inverting the tube several times 4 Incubate at room temperature for 25 minutes capped and protect from light before adding to the samples 5 Setup the luminometer Set the measurement for 1 3 seconds of integration 6 Pipet culture medium samples 10 ul per well in duplicates or in triplicates into a 96 well white opaque or black plate or luminometer tubes Secrete Pair Luminescen
10. ce Assay Kits User Manual 7 Add the GLuc Assay Working Solution from Step 4 100 ul per well or tube to the samples from Step 6 Gently tap the plate tube several times to mix the sample and substrate Do not vortex Note If you have many samples and use 96 well plates we recommend using a multichannel pipette in order to reduce the time between the addition of Assay Working Solution and signal detection For single luminometer tubes do not add the Assay Working Solution to all the tubes samples at one time Instead add the assay working solution to each tube right before its measurement Auto Injector If using an auto Injector equipped luminometer prime the injector with the Assay Working Solution from Step 4 and set the luminometer with the following parameters 100 ul of injection 60 seconds of delay 1 3 seconds of integration and proceed with the measurement without incubations 8 Incubate at room temperature for 1 minute and proceed with the measurement Note Read the plate s within 5 min after the Incubation If using single luminometer tubes make sure the incubation and processing time before the luminescence detection are identical for all samples B Protocol for high sensitivity using GL H buffer Note The wtGLuc activity may not be suitable to measure in Buffer GL H since the activity decay very fast When high throughput screening is pursued in Buffer GL H for high sensitivity we recommend an Auto Injector equipped
11. emperature inverting the tube several times and then vortex for 3 5 Sec Dilute 1 10 in distilled water to make 1x Buffer AP Prepare 100 ul of 1x Buffer AP for each reaction well Duplicates or triplicates for each sample are recommended For example If you have 5 samples in duplicates preparing 1 ml of 1x Buffer AP by diluting 0 1 ml of 10x Buffer AP with 0 9 ml ddH2O Preparing a little bit extra may be helpful to avoid buffer shortage caused by the pipetting error 3 Prepare the SEAP Assay Working Solution e g 10 reactions by adding 10 ul of Substrate AP to 1 ml of 1xBuffer AP Mix well by inverting the tube several times 4 Incubate at room temperature for 5 10 minutes capped and protect from light before adding to the samples 5 Set the luminometer for 1 3 seconds of integration 6 Pipet heated medium samples 10 ul per well in duplicates or triplicates into a 96 well white opaque or black plate or luminometer tubes 7 Add the SEAP Assay Working Solution from Step 4 100 ul per well or tube to the samples from Step 6 Gently tap the plate tube several times to mix the sample and substrate Do not vortex Note If you have many samples and use 96 well plates we recommend using a multichannel pipette in order to reduce the time between the addition of assay working solution and signal detection Auto Injector Since the incubation time is long 5 10 min it is not necessary to use the auto injection for each sam
12. er Manual GLuc is also the brightest luciferase available which generates over 1000 fold higher bioluminescent signal intensity when compared to firefly and Renilla luciferases making it a highly sensitive transcription reporter GLuc is stable over a wide pH range and in the conditioned cell culture medium In vivo GLuc can be detected in blood or urine making it a sensitive tool for real time monitoring of in vivo processes The advantages of Secrete Pair assay kit 1 2 3 4 5 6 No cell lysis Secreted GLuc and SEAP Dual reporter detection Detects GLuc and SEAP Enables transfection normalization for accurate cross sample comparison Highly sensitive and low background GLuc is the brightest luciferase and 1000 fold more sensitive than firefly or Renilla luciferases Easy to eliminate pre accumulated GLuc by changing culture medium Real time study The data is generated quickly and closely resembles real time activities Robust and flexible conditions Two robust buffer conditions are provided for GLuc assays depending on the applications Buffer for stable activity retains more than 90 of signal within the first 10 minutes and extends the half life of light emission to approximately 30 minutes Buffer for higher sensitivity can be used to detect low GLuc expression High throughput compatible Quick and easy assay format High sample number compatible Secrete Pair vs Competitor G
13. from cell culture medium without lysis of the cells A secondary reporter gene SEAP can be used for transfection normalization SEAP is available either on the same vector of GLuc or on a separate vector Secrete Pair Gaussia Luciferase Assay Kit The Gaussia Luciferase Assay kit is designed to analyze the activities of Gaussia Luciferase GLuc only Secreted GLuc can be easily obtained from cell culture medium without lysis of the cells Gaussia luciferase as the reporter gene has strong advantages Gaussia luciferase 185 aa 19 9 kDa is the smallest luciferase It catalyzes the oxidation of the substrate coelenterazine in a reaction that produces light 480 nm E a Gaussia Luciferase aLi TYAS ad HO Q hv 475nm Coelenterazine Coelenteramide Figure 1 The Photo oxidation catalyzed by Gaussia Luciferase Haddock S H D McDougall C M and Case J F The Bioluminescence Web Page http lifesci ucsb edu biolum created 1997 updated 2005 1 Naturally secreted GLuc can be easily collected from cell culture medium without lysis of the cells gt 95 of GLuc is secreted Transfected cells can be kept alive for continuous study such as time course different conditions or other down stream analysis Since the sample collection and activity assay only take minutes the GLuc system enables high throughput screening and also monitors real time activities 4 Secrete Pair Luminescence Assay Kits Us
14. ir Kits 1000 reactions Buffer GL S 10X 1 mix1 pyecotiee Oe cern aaa Glupputr For times pack Slablo tor atleast arasia lusterase i assay kits Buffer GL H 10X imxi a orice 20G pees ene GLuc buffer For high 1 ml x3 p Stable for at least y F aa Gaussia luciferase sensitivity 1 8 ml x6 6 months assay kits 100 ul x4 Dry ice or ice 20 Dual luminescence Substrate GL 100x 100 p x3 pack Stable for at least 8853Y kits GLuc substrate 0 5 i x2 6months Gaussia luciferase i assay kits 1imix1 Dry ice orice 20 C f Buffer AP 10X imix3 pack Stable for at least Dual luminescence SEAP buffer assay kits 1 8 ml x6 6 months 100ulx1 Dry ice orice 20 C Substrate AP 100x Dual luminescence SEAP substrate 100 ul x 3 pack Stable for at least assay kits 0 5 ml x2 6 months 50 ul x 1 Dry ice orice 20 C D llumihesc nce EF1A PG04 Media 50 ul x 2 pack Stable for at least assay kits 50 ul x 2 6 months y The Buffer AP Substrate AP and EF1A PG04 media are only provided in the Dual Luminescence Assay Kits lll Protocol Overview Transfect GLuc SEAP vectors into cells 48 72 hours or after experimental treatment Collect culture medium 10 paced Ny Make 1x Assay Working Solution with luminescent substrate Z 100 pl reaction Setup assays in a 96 well plate black or white or luminometer tubes Measure with a luminometer 4 Secrete Pair Luminescence Assay Kits
15. ple However if using the auto injection prime the injector with the Assay Working Solution from Step 4 and set the luminometer with the following parameters 100 ul of injection 5 10min of delay 1 3 seconds of integration and proceed with the measurement without incubations 8 Incubate at room temperature for 5 10 minutes and proceed with the measurement Note Read the plate s within 5 min after the incubation If using single luminometer tubes make sure the incubation and processing time before the luminescence detection are identical for all samples Vil Signal Normalization only for Secrete Pair dual luminescence assay kit Signal normalization is necessary when comparing GLuc activities of multiple transfected cell samples Using SEAP signal as an internal standard control signal normalization ratio of GLuc and SEAP activities eliminates the impact of transfection efficiency variations and makes the normalized GLuc activities of samples of comparison more accurately reflect the true biological events Calculate the ratio of luminescence intensities RLU Relative Light Unit of the GLuc over SEAP Compare the normalized GLuc activity GLuc SEAP ratio of all samples VIII Important Note The luminescent signals are affected by cell culture media and assay conditions The results should be compared only between samples measured at the same time and using the same medium serum combination Samples collected at different time ma
16. tly tap the plate tube several times to mix the sample and substrate Do not vortex Note If you have many samples and use 96 well plates we recommend using a multichannel pipette in order to reduce the time between the addition of Assay Working Solution and signal detection For single luminometer tubes do not add the Assay Working Solution to all the tubes samples at one time Instead add the assay working solution to each tube right before its measurement Auto Injector If using an auto Injector equipped luminometer prime the injector with the Assay Working Solution from Step 4 and set the luminometer with the following parameters 100 ul of injection 40 seconds of delay 1 2 seconds of integration and proceed with the measurement without incubations 8 Incubate at room temperature for 30 Seconds and proceed with the measurement immediately Note The GLuc activity decays very quickly in Buffer GL H Figure 1 We recommend reading the plate s immediately after the Incubation If using single luminometer tubes make sure the incubation and processing time before the luminescence detection are identical for all samples Secrete Pair Luminescence Assay Kits User Manual VI SEAP Assay Procedure only for Secrete Pair dual luminescence assay kit 1 Aliquot 40 50 ul of each culture medium from GLuc Assay Protocol Step 1 Heat the medium at 65 C for 10 15 min and then place on ice 2 Thaw Buffer AP 10x thoroughly at room t
17. y be stored at 20 C for at least one month without losing the luminescent activity If the GLuc SEAP ratio is too high gt 100 or too low lt 0 01 or either of the luminescent signals RLU is higher than 1 X 10 a sample dilution may be necessary for more stable GLuc and or SEAP luminescent assays Use fresh culture medium for dilution 10 ul of EF1A PG04 media positive control included in the kits normally generate 40 000 80 000 RLU for GLuc and 5 000 10 000 RLU for SEAP in the assays It might vary using different illuminometers Secrete Pair Luminescence Assay Kits User Manual IX References 1 Szent Gyorgyi C et al Proc SPIE 1999 3600 4 11 2 Tannous BA et al Mol Ther 2005 11 435 443 3 Badr CE et al PLoS ONE 2007 2 e571 4 Tannous BA Nat Protoc 2009 4 582 591 X Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Secrete Pair Dual Luminescence Assay Kit or Gaussia Luciferase Assay Kit 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

Secrete-PairTM Gaussia Luciferase Assay Kit

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