Data Sheet Notch Pathway Reporter Kit
Contents
1. 100 So Reporter Activity D o So N 5 4 3 2 14 0 1 2 DAPT log uM 2b Dose response of Notch1AE induced CSL CBF1 RBP Jk reporter activity to DAPT The results are shown as percentage of CSL CBF1 RBP J reporter activity The normalized luciferase activity for Notch1AE transfected cells without DAPT treatment was set at 100 The ICso of DAPT is 0 11 uM References Lu FM et al 1996 Constitutively active human Notch1 binds to the transcription factor CBF 1 and stimulates transcription through a promoter containing a CBF1 responsive element Proc Natl Acad Sci USA 93 11 5663 5667 Kanungo et al 2008 The Notch signaling inhibitor DAPT down regulates cdk5 activity and modulates the distribution of neuronal cytoskeletal proteins J Neurochem 106 2236 OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 1408272. vector 500 ul 20 C Reporter constitutively expressing Renilla 60 ng DNA ul Component B luciferase vector Notch1AE Expression vector for intracellular 250 ul 20 C Component C domain of Notch1 100 ng DNA ul Negative Control Empty expression vector without 250 ul 20 C Expression Notch1 100 ng DNA ul vector Component D These vectors are ready for transient transfection They are NOT SUITBLE for transformation and amplification in bacteria Materials Required but Not Supplied e Mammalian cell line and appropriate cell culture medium e 96 well tissue culture plate or 96 well tissue culture treated white clear bottom assay plate Corning 3610 e Transfection reagent for mammalian cell line We use Lipofectamine 2000 Invitrogen 11668027 However other transfection reagents should work equally well e Opti MEM Reduced Serum Medium Invitrogen 31985 062 e Dual luciferase assay system Dual Glo Luciferase Assay System Promega E2920 This system assays cells directly in growth medium It can be used with any luminometer Automated injectors are not required OR Dual Luciferase Reporter Assay System Promega E1910 This system requires a cell lysis step It is ideal for luminometers with automated injectors e Luminometer OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 86
3. 6044 Cornerstone Court W Ste E Bi i San Diego CA 92121 Tel 1 858 829 3082 lOSCIENCE Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet Notch Pathway Reporter Kit Catalog 60509 Background The Notch signaling pathway controls cell fate decisions in vertebrate and invertebrate tissues NOTCH signaling is triggered through the binding of a transmembrane ligand to Notch transmembrane receptor NOTCH1 NOTCH2 NOTCH3 NOTCH4 on a neighboring cell This results in proteolytic cleavage of the NOTCH receptor releasing the constitutively active intracellular domain of NOTCH NICD NICD translocates to the nucleus and associates with transcription factors CSL CBF1 RBPJk Suppressor of Hairless Lag 1 and coactivator Mastermind to turn on transcription of Notch responsive genes Description Notch Pathway Reporter kit is designed for monitoring the activity of the Notch signaling pathway in cultured cells The kit contains a transfection ready expression vector for NOTCH1 that has a deletion of the entire extracellular domain Notch1AE Inside the cells the NOTCH1 AE can be cleaved by y secretase and active NOTCH1 NICD is released into the nucleus The kit also contains CSL CBF1 RBP Jk luciferase reporter vector which is a Notch pathway responsive reporter This reporter contains the firefly luciferase gene under the control of multimerized CSL responsive elements upstream of a minimal promoter The CSL CBF1 RBP Jx reporter i
4. 94 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 140827 6044 Cornerstone Court W Ste E 6 Bi p San Diego CA 92121 Tel 1 858 829 3082 lOSCIENCE Fax 1 858 481 8694 Email info bpsbioscience com Generalized Transfection and Assay Protocols The following procedure is designed to transfect the reporter into HEK293 cells using Lipofectamine 2000 in a 96 well format To transfect cells in different tissue culture formats adjust the amounts of reagents and cell number in proportion to the relative surface area If using a transfection reagent other than Lipofectamine 2000 follow the manufacturer s transfection protocol Transfection condition should be optimized according to the cell type and study requirements All amounts and volumes in the following setup are given on a per well basis 1 One day before transfection seed cells at a density of 30 000 cells per well in 100 ul of growth medium so that cells will be 90 confluent at the time of transfection 2 Next day for each well prepare complexes as follows a Dilute DNA mixtures in 15 ul of Opti MEM medium antibiotic free Mix gently Depending upon the experimental design the DNA mixtures may be any of the following combinations e 1 ul of Reporter component A in this experiment the control transfection is 1 ul of Negative Control Reporter component B e 1 ul of Reporter component A experimental vecto
5. bpsbioscience com 140827 6044 Cornerstone Court W Ste E Bi i San Diego CA 92121 Tel 1 858 829 3082 lOSCIENCE Fax 1 858 481 8694 Email info bpsbioscience com 3 Add the 30 ul of complexes to each well containing cells and medium Mix gently by tapping the plate 4 Incubate cells at 37 C in a CO incubator After 24 hours of transfection change medium to fresh growth medium 48 hours after transfection perform the dual luciferase assay following the manufacturer s protocol To study the effect of activators inhibitors on the Notch pathway treat the cells with tested activator inhibitor after 6 hours or 24 hours of transfection Perform dual luciferase assay 48 hours after transfection Sample protocol to determine the effect of NOTCH 1 on the CSL CBF1 RBP Jk reporter in HEK293 cells 1 One day before transfection seed HEK293 cells at a density of 30 000 cells per well into white clear bottom 96 well plate in 100 ul of growth medium MEM EBSS Hyclone SH30024 01 10 FBS 1 non essential amino acids 1 mM Na pyruvate 1 Pen Strep Incubate cells at 37 C in a CO incubator overnight Next day transfect 1 ul of CSL CBF1 RBP Jx luciferase reporter component A with 0 5 ul of Notch1AE component C or negative control expression vector component D into cells following the procedure in Generalized Transfection and Assay Protocols After 24 hours of transfection change medium to 50 ul of fresh
6. er en CSL CBF1 RBP J x reporter Negative control reporter Sample protocol to determine the effect of antagonists of Notch signaling pathway on Notch1AE induced CSL CBF1 RBP Jk reporter activity in HEK293 cells 1 One day before transfection seed HEK293 cells at a density of 30 000 cells in 100 ul of growth medium into each well of a white clear bottom 96 well plate Incubate cells at 37 C in a CO incubator overnight 2 Next day transfect 1 ul of CSL CBF1 RBP Jk luciferase reporter component A with 0 5 ul of NotchiAE component C into cells following the procedure in Generalized Transfection and Assay Protocols 3 After 24 hours of transfection treat transfected cells with the Notch pathway inhibitor DAPT y secretase inhibitor in 50 ul of fresh growth medium Add 50 ul of growth medium to cell free control wells for determining background luminescence Set up each treatment in at least triplicate Incubate cells at 37 C in a CO incubator for 24 hours 4 After 48 hours of transfection perform dual luciferase assay using Dual Glo Luciferase Assay System Add 50 ul of Luciferase reagent per well and rock at room temperature for 15 minutes then measure firefly luminescence using a luminometer Add 50 ul of Stop amp OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbio
7. growth medium Add 50 ul of growth medium to cell free control wells for determining background luminescence After 48 hours of transfection perform dual luciferase assay using Dual Glo Luciferase Assay System Promega E2920 Add 50 ul of Luciferase reagent per well and rock at room temperature for 15 minutes then measure firefly luminescence using a luminometer Add 50 ul of Stop amp Glo reagent per well Rock at room temperature for 15 minutes and measure Renilla luminescence To obtain the normalized luciferase activity for CSL CBF1 RBP Jk reporter subtract background luminescence then calculate the ratio of firefly luminescence from CSL CBF1 RBP Jk reporter to Renilla luminescence from the control Renilla luciferase vector OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 140827 6044 Cornerstone Court W Ste E 6 Bi g San Diego CA 92121 Tel 1 858 829 3082 lOSCIEMNCE Fax 1 858 481 8694 Email info bpsbioscience com Figure 1 Notch1AE induced the expression of CSL CBF1 RBP Jx reporter The results are shown as normalized CSL CBF1 RBP Jk luciferase reporter activity 1 4 12 1 0 8 0 6 0 4 0 2 0 Normalized luciferase activity cant ecto patah se cart acto pares a a ree yee
8. r expressing gene of interest such as component C in this experiment the control transfections are 1 ul of Reporter component A negative control expression vector Such as component D 1 ul of Negative Control Reporter component B experimental vector expressing gene of interest and 1 ul of Negative Control Reporter component B negative control expression vector e 1 ul of Reporter component A specific siRNA in this experiment the control transfections are 1 ul of Reporter component A negative control siRNA 1 ul of Negative Control Reporter component B specific siRNA and 1 ul of Negative Control Reporter component B negative control siRNA Note we recommend setting up each condition in at least triplicate and prepare transfection cocktail for multiple wells b Mix Lipofectamine 2000 gently before use then dilute 0 35 ul of Lipofectamine 2000 in 15 ul of Opti MEM medium antibiotic free Incubate for 5 minutes at room temperature Note Prepare this dilution cocktail in volumes sufficient for the whole experiment b After the 5 minute incubation combine the diluted DNA with diluted Lipofectamine 2000 Mix gently and incubate for 25 minutes at room temperature OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www
9. s premixed with constitutively expressing Renilla sea pansy luciferase vector which serves as an internal positive control for transfection efficiency The kit also includes a non inducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as a negative control The non inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter but without any additional response elements The negative control is critical for determining pathway specific effects and background luciferase activity Application e Monitor Notch signaling pathway activity e Screen activators or inhibitors of Notch signaling pathway e Study effects of RNAi or gene overexpression on the activity of Notch pathway OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 140827 6044 Cornerstone Court W Ste E 6 Bi p San Diego CA 92121 Tel 1 858 829 3082 lOSCIENCE Fax 1 858 481 8694 Email info bpsbioscience com Components Component Specification Amount Storage Reporter CSL CBF1 RBP Jk luciferase 500 ul 20 C Component A reporter vector constitutively 60 ng DNA ul expressing Renilla luciferase vector Negative Control Non inducible luciferase
10. science com Please visit our website at www bpsbioscience com 140827 6044 Cornerstone Court W Ste E 6 Bi g San Diego CA 92121 Tel 1 858 829 3082 lOSCIEMNCE Fax 1 858 481 8694 Email info bpsbioscience com Glo reagent per well Rock at room temperature for 15 minutes and measure Renilla luminescence 5 To obtain the normalized luciferase activity for CSL CBF1 RBP Jk reporter subtract background luminescence then calculate the ratio of firefly luminescence from CSL CBF1 RBP Jk reporter to Renilla luminescence from the control Renilla luciferase vector Figure 2 Inhibition of Notch1AE induced CSL CBF1 RBP Jk reporter activity by Notch pathway inhibitor DAPT y secretase inhibitor ie 0 25 N gt u uo 0 05 Normalized luciferase activity DAPT s 10 uM 7 10 uM Control Notch1 AE expression vector 2a DAPT completely blocks Notch1AE induced CSL CBF1 RBP uJk reporter activity The results are shown as normalized CSL CBF1 RBP Jx luciferase reporter activity OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by Phone 1 858 829 3082 Fax 1 858 481 8694 Or you can Email us at info bpsbioscience com Please visit our website at www bpsbioscience com 140827 6044 Cornerstone Court W Ste E Bi F San Diego CA 92121 Tel 1 858 829 3082 lOSCIENCE Fax 1 858 481 8694 Email info bpsbioscience com IC50 0 11 uM
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