User Manual - Biomol GmbH
Contents
1. E2F E2F binding element E2F DP1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 PKC Ca NFAT response element NFAT TGFB SMAD response element SMAD2 SMAD3 SMAD4 Myc Max E box binding element Myc Max Technical Support 888 503 3187 US 15 301 682 9200 Cignal Finder 10 Pathway Reporter Arrays plate format Part 1036A Version 1 3 9 2 2008 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com2. ml Penicillin and 100 ug ml Streptomycin instead of growth medium in step 9 and treating the transfected cells with 3 or 4 different concentrations of small chemicals for 6 to 24 hours prior to assay development Technical Support 888 503 3187 US 301 682 9200 13 Appendix Cignal Finder 10 Pathway Reporter Arrays Cignal Finder Cancer 10 Pathway Reporter Array Tube Format CCA 001L Plate Format CCA 101L Version 1 3 Pathway Transcriptional Regulatory Element TRE Transcription Factor Wnt TCF LEF response element TCF LEF Notch RBP Jx binding element RBP Jx p53 DNA Damage p53 response element p53 TGFB SMAD response element SMAD2 SMAD3 SMAD4 Cell Cycle pRb E2F E2F binding element E2F DP1 NFkB NF B binding element NFkB Myc Max E box binding element Myc Max Hypoxia HIF response element Hypoxia inducible factor 1 HIF 1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 Cignal Finder Immune Response 10 Pathway Reporter Array Tube Format CCA 002L Plate Format CCA 102L Pathway Transcriptional Regulatory Element TRE Transcription Factor NF B NF B binding element NF B PKC Ca NFAT response element NFAT Type Interferon Interferon stimulated response element ISRE STAT1 STAT2 Interferon Gamma Interferon gamma activation sequence GAS STAT1 STAT1 MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP
3. ul of Opti MEM sufficient for 108 transfections Mix gently by inverting tube slowly and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 50 ul of diluted SureFECT into each well containing 50 ul of the diluted nucleic acids 1 1 ratio 4 Mix by gently tapping the sides of the plate for at least 30 seconds and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 6 x 10 cells ml in Opti MEM containing 10 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed mix the cell suspension by several inversions of the tube containing the cells or by gentle pipeting of the cell suspension 7 Add 50 ul of prepared cell suspension 3 x 104 cells in Opti MEM containing 10 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume in each well of 150 ul Mix gently by
4. 1 binding element AP 1 TGFB SMAD response element SMAD2 SMAD3 SMAD4 cAMP PKA cAMP regulatory element CRE CREB C EBP C EBP binding element C EBP Glucocorticoid Receptor Glucocorticoid response element GRE Glucocorticoid Receptor GR Technical Support 888 503 3187 US 14 301 682 9200 Version 1 3 Cignal Finder 10 Pathway Reporter Arrays continued Cignal Finder Development 10 Pathway Reporter Array Tube Format CCA 003L Plate Format CCA 103L Pathway Transcriptional Regulatory Element TRE Transcription Factor Notch RBP J binding element RBP Jic Wnt TCF LEF response element TCF LEF Myc Max E box binding element Myc Max NFkB NF B binding element NFkB TGFB SMAD response element SMAD2 SMAD3 SMAD4 Cell Cycle pRb E2F E2F binding element E2F DP1 C EBP C EBP binding element C EBP cAMP PKA cAMP regulatory element CRE CREB MAPK ERK Serum response element SRE Elk 1 SRF MAPK JNK AP 1 binding element AP 1 Cignal Finder Toxicity 10 Pathway Reporter Array Tube Format CCA 004L Plate Format CCA 104L Pathway Transcriptional Regulatory Element TRE Transcription Factor p53 DNA Damage p53 response element p53 Hypoxia HIF response element Hypoxia inducible factor 1 HIF 1 NF B NF B binding element NF B Glucocorticoid Receptor Glucocorticoid response element GRE Glucocorticoid Receptor GR Cell Cycle pRb
5. IMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of Cignal Finder Reporter Arrays includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any constructs to modify kit components for resale or to manufacture commercial products without written approval of SABiosciences Bioscience Corporation No other license expressed implied or by estoppel is granted NOTICE TO PURCHASER II The Dual Luciferase Reporter Assay is a trademark of Promega Corporation SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA Technical Support support SABiosciences com www SABiosciences com 2 CONTENTS l Introduction Il Product Contents and Descriptions Ill Additional M
6. SABiosciences REET Waidmanns tr 35 O 22769 Hamburg b info biomol de iomo www biomol de Phone 49 40 8532600 or 0800 2466651 D U S e r M a n u a Fax 49 40 85326022 or 0800 2466652 D Cignal Finder 10 Pathway Reporter Arrays plate format Cell Based Multi Pathway Activity Assays See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1036A Version 1 3 9 2 2008 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Cignal Finder 10 Pathway Reporter Arrays plate format Cell Based Multi Pathway Activity Assays User Manual For Catalog Numbers CCA 1XXL onan z i i zeroes CY biomol www biomol de Ordering and Technical Service Contact Information Phone 49 40 8532600_ or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www SABiosciences com e E MAIL order SABiosciences com to place an order support SABiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com L
7. al Finder Toxicity 10 Pathway Reporter Array CCA 104L Technical Support 888 503 3187 US 301 682 9200 4 Version 1 3 gt EE a Transcription Negative Factor 3 Control Add SureFECT Transfection J Reagent amp Test Nucleic Acids Seed Cells for Reverse Transfection J Treat amp Analyze Phenotype with a Cell based Assay Dual luciferase Figure 1 Overview of Cignal Finder Reporter Array plate format Process Technical Support 888 503 3187 US 301 682 9200 5 ll Product Contents and Descriptions Version 1 3 A Cignal Finder 10 Pathway Reporter Array Contents Table 1 Cignal Finder Reporter Array plate format Specifications expressing Renilla luciferase construct 20 1 1 eee Total DNA in Component Specification each well A mixture of an inducible transcription factor Each of the responsive firefly luciferase reporter and 200 ng 10 Reporter constitutively expressing Renilla construct Assays 20 1 Negative A mixture of non inducible firefly luciferase control reporter and constitutively expressing 200 ng Renilla construct 20 1 A mixture of a constitutively expressing GFP Positive construct constitutively expressing firefly 200 ng control luciferase construct and constitutively NOTE All constructs are transfection grade and are ready for transient transfection These constructs are specifically designed to inhibit transformation and are NOT MEANT for i
8. al luciferase reporter encodes for the mammalian codon optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly degrade the destabilized form of the firefly luciferase protein and hence the background luciferase activity noise level is greatly reduced Due to low background activity the magnitude of the response that can be measured signal to noise ratio as well as the speed of measuring changes in transcription are enhanced The Cignal dual luciferase reporter assays provide outstanding reproducibility sensitivity specificity and signal to noise ratio They are extremely useful assays for carrying out quantitative pathway regulation studies Benefits of Cignal Finder 10 Pathway Reporter Arrays e BIOLOGICAL PROCESS FOCUSED Profile the changes in the activities of ten signaling pathways relevant to a specific biological process e HIGH PERFORMANCE Dual luciferase assay provides high sensitivity specificity and reproducibility e FLEXIBILITY AND CONVENIENCE Utilize a straightforward reverse transfection procedure with your favorite cell lines to rapidly generate valuable mechanism of action data Available Cignal Finder 10 Pathway Reporter Arrays plate format Product Name Catalog Number Cignal Finder Cancer 10 Pathway Reporter Array CCA 101L Cignal Finder Immune Response 10 Pathway Reporter Array CCA 102L Cignal Finder Development 10 Pathway Reporter Array CCA 103L Cign
9. aterials Required IV Protocol A Before you begin B Protocol Appendix Cignal Finder 10 Pathway Array Product Descriptions Technical Support 888 503 3187 US 301 682 9200 3 10 10 12 14 Version 1 3 Introduction The Cignal Finder 10 Pathway Reporter Arrays enable you to pinpoint the pathways regulated by the gene products or chemical compounds being studied in your laboratory The Cignal Finder Arrays consist of 10 dual luciferase reporter assays and are designed for use in one of four research areas The targeted research areas are cancer immunology development and toxicology In this era of post genomics life science research many labs are investigating how diverse signal transduction pathways function on their own and in combination within the cell The Cignal Finder Arrays equip life science researchers to carry out such studies with speed and confidence These arrays are delivered in 96 well plates including important negative and positive controls Each of the twelve columns of the 96 well plate contains eight wells with a pathway focused reporter or control dried down in all eight wells The reporter assays are used right out of the box for the reverse transfection of the reporter assays into your cell lines of interest The Cignal Finder Reporter Arrays are valuable tools for progressing from the identification of genes proteins or small molecules to understanding their function Each pathway focused du
10. nd cells to appropriate density Immediately seed 50 uL of suspended cells to each well Replace growth medium after 16 24 hours of transfection DAY 1 Technical Support 888 503 3187 US 301 682 9200 11 Version 1 3 B PROTOCOL The following protocol is designed to reverse transfect an adherent cell line HEK293 using SureFECT Transfection Reagent Cat No SA 01 The Cignal Finder Array plates work well with transfection reagents from other vendors f you are using a transfection reagent other than SureFECT follow their manufacturers protocol for optimizing transfection This is just a general guideline the optimal conditions amounts should be optimized according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium 1 Add 50 ul of Opti MEM to each well of the Cignal Finder Array plate avoid using DMEM Resuspend the reporter assay constructs by gently tapping the side of the plate while slightly rocking the plate back and forth then left to right five times each and incubate it for 5 min at room temperature 2 We typically use 0 3 ul of SureFECT in 50 ul of Opti MEM per well for each individual transfection In order to prepare sufficient SureFECT for an entire 96 well plate we recommend diluting 32 4 ul of SureFECT into 5400
11. ndition The response rate in the Cignal Reporter Assay depends on the assay conditions conditions of the experimental treatment To obtain maximum response given by any stimulus perform dosing and time course studies The optimal amount of stimulus and the time of treatment must be obtained empirically for each experiment see different protocols for our recommendations Important recommendations for best results A Perform all transfections in triplicate to minimize variability among treatment groups B Include positive and negative controls in each experiment to obtain reliable results C Use low passage cells that are actively growing and are greater than 90 viable for maximal transfection efficiencies D Do not add antibiotics to media during transfection as this may cause cell death Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment F Serum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 5 in the assay medium during the experimental treatment to minimize these serum effects m Technical Support 888 503 3187 US 301 682 9200 10 6 R Version 1 3 Transfection Protocols In order to use the Cignal Finder 10 Pathway Arrays in the plate format a reverse transfection method must be employed This approach involves seeding the cell line of interest onto the transfec
12. ntroduction and amplification in bacteria Each kit also includes a white self adhesive sealing tape for each plate included in the kit This tape should be affixed to the bottom of each plate immediately prior to reading the plate in a plate reading luminometer in order to maximize the signal to noise ratio of each reading B Description of Individual Cignal Reporter Assays Each Cignal Reporter Assay Kit includes the following components 1 Reporter Each reporter is a mixture of an inducible transcription factor responsive construct and constitutively expressing Renilla luciferase construct 20 1 The inducible transcription factor responsive construct encodes the firefly luciferase reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE Figure 2A This construct monitors both increases and decreases in the activity of a key transcription factor which is a downstream target of a specific signaling pathway The constitutively expressing Renilla construct encodes the Renilla luciferase reporter gene under the control of a CMV immediate early enhancer promoter Figure 2B and acts as an internal control for normalizing transfection efficiencies and monitoring cell viability It is also useful to confirm transfection and to verify active luciferase in the transfected culture Technical Support 888 503 3187 US 6 301 682 9200 2 Ve
13. onal activator activities in the cell line used will determine the sensitivity of the assay A cell line should be selected based on the functionality of the signal transduction pathway under investigation as well as for the transfectability of the cell line see below Transfection reagent selection We recommend the use of SureFECT SABiosciences Cat No SA 01 as a transfection reagent The Cignal Reporter Assay however also performs equally well with other transfection reagents such as Lipofectamine 2000 Invitrogen Cat No 11668 027 or FUGENE 6 Roche Cat No 1815091 When using alternative transfection reagents please refer to the manufacturer s instructions on the use of those reagents Optimization of transfection conditions The sensitivity of the Cignal Reporter Assay depends on the transfection efficiency The transfection efficiency in turn primarily depends upon cell line used Therefore it is very important to optimize the transfection conditions for each cell type under study Variables to consider when optimizing the transfection conditions include cell density cell viability amount of DNA ratio of DNA to transfection reagent transfection complex formation time and transfection incubation time see the detailed protocols for our recommendations The positive control construct included with each Cignal Reporter Assay can be used for determining the optimal transfection conditions Optimization of assay co
14. pport 888 503 3187 US 301 682 9200 8 Version 1 3 Additional Materials Required Mammalian cell line cultured in the appropriate growth medium Cell culture medium and standard cell culture supplies Multi channel pipettor and pipettor reservoirs Transfection reagent We recommend SureFECT SABiosciences Cat No SA 01 however other transfection reagents work equally well Polystyrene test tubes BD FALCON Cat 352099 Opti MEM Reduced Serum Medium Invitrogen Cat No 31985 062 Fetal bovine serum FBS Non essential amino acids NEAA Invitrogen Cat No 11140 050 Penicillin Streptomycin Hemacytometer Dual Luciferase Assay System o Dual Luciferase Reporter Assay System Promega Cat No E1910 This system requires cell lysis and is well suited for the rapid quantitation of both luciferase reporters when using luminometers with reagent auto injectors o Dual Glo Luciferase Assay System Promega Cat No E2920 This system is used to assay for both luciferase reporters on intact cells in growth medium This system can be used with any luminometer including those without reagent auto injectors Luminometer Technical Support 888 503 3187 US 301 682 9200 9 Version 1 3 IV Protocol A Before you begin 1 Cell line selection The Cignal Reporter Assay may be used with various mammalian cell lines Cell lines show a great deal of variation in the levels of signaling proteins The transcripti
15. rocking the plate back and forth then left to right Do not move the plate in a circular motion as this may cause the cells to preferentially sediment around the edges of each well Technical Support 888 503 3187 US 301 682 9200 12 Version 1 3 8 Incubate cells at 37 C in a 5 COs incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 Carry out the luciferase assay using either the Dual Luciferase Reporter Assay System or Dual Glo Luciferase Assay System from Promega Follow the manufacturer s protocol for developing the assay Please see specific recommendations in the Important Notes section below for some general recommendations on when to carry out the luciferase assays for different types of studies Each Cignal Finder Array plate comes along with a white self adhesive sticker which should be attached to the bottom of the plate before reading the luciferase activity Using the sticker to cover the optical bottom of the 96 well plate helps to maximize the signal to noise ratio of each reading Important Notes Here we have included some general recommendations for different experimental designs 1 To determine the effect of siIRNA shRNA on 10 different cell signaling pathways we recommend doing transient co transfection of siIRNA shRNA and reporter constructs For thi
16. rsion 1 3 Negative control The negative control is a mixture of non inducible reporter construct and constitutively expressing Renilla luciferase construct 20 1 The non inducible reporter construct encodes firefly luciferase under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 2C The negative control is critical to identifying specific effects and determining background reporter activity Positive control The positive control is a constitutively expressing GFP construct Figure 2D pre mixed with a constitutively expressing firefly luciferase construct Figure 2E and a constitutively expressing Renilla luciferase construct Figure 2B 40 1 1 The positive control is necessary for visual confirmation of transfection It is also useful for transfection optimization studies The expression of the GFP from the positive control construct can be monitored by fluorescence microscopy using an excitation filter of 470 20 nm 470 40 nm and an emission filter of 515 nm long pass Tandem repeats TATA box A of TREs y Firefly Luc CMV immediate early enhancer promoter E TATA box C ii Firefly Luc D CMV immediate early Lal enhancer promoter CMV immedi Figure 2 Schematic representation of constructs involved in the Cignal Reporter Assay A The inducible transcription factor responsive construct expressing firefly luciferase B The consti
17. s one can add 2 pmol of siRNA or 200 ng of shRNA plasmid to the resuspended reporter construct in step 7 of the protocol The luciferase assay can be developed 48 72 hours after the co transfection Please remember to include negative control siRNA shRNA to assist in the interpretation of your results 2 To determine the effect of cDNA overexpression on 10 different cell signaling pathways we recommend doing the transient co transfection of experimental vector and reporter constructs For this one can add 100 200 ng of experimental vector to the resuspended reporter construct in step 1 of the protocol The luciferase assay can be developed 36 48 hours after the co transfection Please remember to include negative control vector empty vector to assist in the interpretation of your results 3 To determine the effect of recombinant protein or small peptide on 10 different cell signaling pathways we recommend changing the cell medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin instead of growth medium in step 9 and treating the transfected cells with 3 or 4 different concentrations of recombinant protein or small peptide for 6 to 24 hours prior to assay development 4 To determine the effect of small chemicals on 10 different cell signaling pathways we recommend changing the cell medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U
18. tion complexes in a one day procedure This is in contrast to traditional transfection methods in which cells are seeded on the first day of the experiment and transfection complexes are added to the cells the following day The SureFECT transfection reagent has been specifically developed as a reverse transfection reagent Optimized reverse transfection protocols using the SureFECT transfection reagent are described throughout the Cignal Finder Reporter Arrays User Manual Utilizing reverse transfection procedures results in both a time savings as well as improved reproducibility when compared to traditional forward transfection methods Conditions for using transfection reagents from other vendors in reverse transfection protocols may also be developed This will require initial process optimization studies Below is a general protocol overview for reverse transfection of the Cignal Finder 10 Pathway Reporter Arrays everse Transfection Protocol Overview 1 DAY PROCEDURE Reverse Transfection 96 well Cell Culture Plate LEE Add SureFECT Transfection Reagent and Test Nucleic Acids 900 Seed Cells for Reverse Transfection D Add 50 uL of Opti MEM to each well of Cignal Finder array plate to resuspend reporter constructs Dilute SureFECT into Opti MEM Add 50 uL of diluted SureFECT to 50 uL of resuspended reporter constructs mix well and incubate at room temperature for 20 minutes Trypsinize if necessary count and suspe
19. tutively expressing Renilla luciferase construct C The non inducible firefly luciferase reporter construct D The constitutively expressing GFP construct and E The constitutively expressing firefly luciferase construct Technical Support 888 503 3187 US 301 682 9200 7 Version 1 3 IMPORTANT NOTE There are a few reports in the literature of the CMV regulatory element being activated by certain stimuli see below We recommend that you confirm that the stimulus used in each Cignal reporter assay does not induce the CMV regulatory element in order to confirm that the CMV Renilla construct is the appropriate normalization construct for your experiment This can be done empirically by testing the impact of your stimulus on the Cignal positive control reporters which are each under the control of the CMV enhancer promoter cassette If your stimulus is one of the very few reported activators of the CMV regulatory element we advise using an alternative reporter as an internal control e W Bruening B Giasson W Mushynski and H D Durham 1998 Nucleic Acids Research 26 2 486 489 Activation of stress activated MAP protein kinases up regulates expression of transgenes driven by the cytomegalovirus immediate early promoter e Madhu S Malo Moushumi Mozumder Alexander Chen Golam Mostafa Xiao Bo Zhang Richard A Hodin 2006 Analytical Biochemistry 350 307 309 pFRL7 An ideal vector for eukaryotic promoter analysis Technical Su
Download Pdf Manuals
Related Search