Data Sheet - BPSBioscience.com
Contents
1. control of a minimal promoter without any additional response elements The negative control is critical to determining pathway specific effects and background luciferase activity Applications e Monitor Wnt signaling pathway activity e Screen activators or inhibitors of Wnt B catenin signaling pathway e Study effects of RNAi or gene overexpression on the activity of Wnt 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 San Diego CA 92121 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Components Component Specification Amount Storage Reporter TCF LEF luciferase 500 ul 20 C Component A reporter vector 60 ng DNA ul constitutively expressing Renilla luciferase vector Negative Contol Non inducible 500 ul 20 C Reporter luciferase vector 60 ng DNA ul Component B constitutively expressing Renilla luciferase vector These vectors are ready for transient transfection They are NOT MEANT for transformation and amplication 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 c2. 0 ul of fresh growth medium Incubate cells at 37 in a CO2 incubator for 16 hours For wells without IWR 1 endo treat cells with LiCl only 4 After 40 hours of transfection add mouse Wnt3a final concentration 40 ng ml in 5ul of growth medium to stimulated wells cells treated with Wnt8a LiCl and with or without IWR 1 endo add 5ul of growth medium to the unstimulated control wells cells treated with LiCl only for determining the basal activity add 55ul of growth medium to cell free control wells for determining background luminescence Set up each treatment in at least triplicate 5 Incubate at 37 in a CO2 incubator for 5 6 hours 6 Perform dual luciferase assay using Dual Glo Luciferase Assay System Add 55 ul of Luciferase reagent per well and rocking at room temperature for 15 minutes and measure firefly luminescence using a luminometer Add 55 ul of Stop amp Glo reagent per well and rocking at room temperature for 15 minutes and measure Renilla luminescence 7 To obtain the normalized luciferase activity of TCF LEF reporter subtract background luminescence then calculate the ratio of firefly luminescence from the TCF LEF reporter to Renilla luminescence from the control Renilla luciferase vector Figure 2 Inhibition of Wnt3a induced TCF LEF reporter activity by IWR 1 endo Figure 2a IWR 1 endo completely blocked Wnt3a induced TCF LEF reporter activity 1 2 1 0 8 0 6 0 4 0 2 Normalized Luci
3. 6044 Cornerstone Court W Ste E Bi San Diego CA 92121 Tel 1 858 829 3082 lOSCICNCE Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet TCF LEF Reporter Kit Wnt B catenin signaling pathway Catalog 60500 Background The Wnt B catenin signaling pathway controls a large and diverse set of cell fate decisions in embryonic development adult organ maintenance and disease Wnt proteins bind to receptors on the cell surface initiating a signaling cascade that leads to stabilization and nuclear translocation of B catenin B catenin then binds to TCF LEF transcription factors in the nucleus leading to transcription and expression of Wnt responsive genes Description The TCF LEF Reporter kit is designed for monitoring the activity of Wnt B catenin signaling pathway in the cultured cells The kit contains transfection ready TCF LEF luciferase reporter vector which is a Wnt pathway responsive reporter This reporter contains a firefly luciferase gene under the control of multimerized TCF LEF responsive element located upstream of a minimal promoter The TCF LEF reporter is premixed with constitutively expressing Renilla luciferase vector that serves as internal control for transfection efficiency The kit also includes a non inducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as negative control The non inducible luciferase vector contains a firefly luciferase gene under the
4. ferase Activity 0 LiCl Wnt3a IWR 1 endo 10 uM 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 San Diego CA 92121 M 1 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Figure 2b Dose response of Wnt3a induced TCF LEF reporter activity to IWR 1 endo The results were shown as percentage of TCF LEF reporter activity The normalized luciferase activity for cells stimulated with Wnt3a in the absence of IWR 1 endo was set at 100 The IC50 of IWR 1 endo is 0 05 uM IC50 0 05 uM o od O oo Reporter Activity A o O O N o oOo 4 3 2 4 0 1 2 IWR 1 endo Log uM Reference Chen B et al 2009 Small molecule mediated disruption of Wnt dependent signaling in tissue regeneration and cancer Nature Chemical Biology 5 2 100 107 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
5. iferase assay using Dual Glo Luciferase Assay System Add 55 ul of Luciferase reagent per well and rocking at room temperature for 15 minutes and measure firefly luminescence using a luminometer Add 55 ul of Stop amp Glo reagent per well and rocking at room temperature for 15 minutes and measure Renilla luminescence 7 To obtain the normalized luciferase activity for TCF LEF reporter subtract background luminescence then calculate the ratio of firefly luminescence from the TCF LEF 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 San Diego CA 92121 M 1 Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Figure 1 Dose response of TCF LEF reporter activity to mouse Wnt3a The results were shown as fold induction of normalized TCF LEF luciferase reporter activity Fold induction was determined by comparing values against the mean value for control cells without Wnt8a treatment The EC50 of mWnt8a is 16 ng ml 10 0 EC50 16 ng ml 5 75 g 5 0 D L 25 0 0 11 10 9 8 7 6 5 Wnt3a Log g ml Sample protocol to determine the effect of antagonists of Wn
6. ioscience com Sample protocol to determine the dose response of HEK293 cells transfected with TCF LEF reporter to mouse Wnt3a Additional materials required in this experiment setup e LiCl Sigma L7026 e Mouse Wnt3a R amp D Systems 1324 WN e HEK293 growth medium MEM EBSS with L glutamine Hyclone SH30024 01 10 FBS 1 non essential amino acid 1mM Na pyruvate 1 Pen Strep e 96 well tissue culture treated white clear bottom assay plate Corning 3610 e Dual Glo Luciferase Assay System Promega E2920 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 Incubate cells at 37 in a CO2 incubator for overnight 2 Next day transfect 1 ul of TCF LEF luciferase reporter component A into cells fowllowing the procedure in Generalized Transfection and Assay Protocols 3 After 24 hours of transfection treat transfected cells with LiCl 1 0mM in 50 ul of fresh growth medium Incubate cells at 37 in a CO2 incubator for 16 hours 4 After 40 hours of transfection add threefold serial dilution of mouse Wnt3a in 5ul of growth medium to stimulated wells add 5u of growth medium to unstimulated control wells add 55ul of growth medium to cell free control wells for determining background luminescence Set up each treatment in at least triplicate 5 Incubate at 37 in a CO2 incubator for 5 6 hours 6 Perform dual luc
7. ourt W Ste E Bi San Diego CA 92121 Tel 1 858 829 3082 IOSCIENCEe Fax 1 858 481 8694 Email info bpsbioscience com All amounts and volumes in the following setup are given on a per well basis 1 One day before transfection seed HEK293 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 following combinations e 1 ul of Reporter component A in this experiment the control transfection is 1 pl of Negative Control Reporter component B e 1 pl of Reporter component A experimental vector expressing gene of interest in this experiment the control transfection is 1 wl of Reporter component A negative control expression vector 1 yl of Negative Control Reporter component B experimental vector expressing gene of interest and 1 wl of Negative Control Reporter component B negative control expression vector e 1 ul of Reporter component A specific siRNA in this experiment the control transfection is 1 ul of Reporter component A negative control siRNA 1 ul of Negative Control Reporter component B specific siRNA and 1 pl of Negative Control Reporter component B negative control siRNA Note we recommend
8. setting up at least triplicates for each condition 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 c After the 5 minute incubation combine the diluted DNA with diluted Lipofectamine 2000 Mix gently and incubate for 25 minutes at room temperature 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 CO2 incubator After 24 hours of transfection change medium to fresh growth medium 48 hours after transfection perform dual luciferase assay following manufacturer s protocol To study the effect of activators inhibitors on the Wnt pathway treat the cells with tested activator inhibitor after 24 hours or 42 hours of transfection Perform dual luciferase assay 48 hours after transfection 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 San Diego CA 92121 Tel 1 858 829 3082 IOSCIENCEe Fax 1 858 481 8694 Email info bpsb
9. t signaling pathway on Wnt3a induced TCF LEF reporter activity in HEK293 cells Additional materials required in this experiment setup e IWR 1 endo Santa Cruz biotechnology sc 295215 inhibitor of Wnt pathway e LiCl Sigma L7026 e Mouse Wnt3a R amp D Systems 1324 WN e HEK293 growth medium MEM EBSS with L glutamine Hyclone SH30024 01 10 FBS 1 non essential amino acid 1mM Na pyruvate 1 Pen Strep e 96 well tissue culture treated white clear bottom assay plate Corning 3610 e Dual Glo Luciferase Assay System Promega E2920 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 Incubate cells at 37 in a CO2 incubator for overnight 2 Next day transfect 1 ul of TCF LEF luciferase reporter component A into cells fowllowing the procedure in Generalized Transfection and Assay Protocols 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 o San Diego CA 92121 Bioscience Tel 1 858 829 3082 Fax 1 858 481 8694 Email info bpsbioscience com 3 After 24 hours of transfection treat transfected cells with threefold serial dilution of IWR 1 endo plus LiCl 10mM in 5
10. ulture 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 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 assay 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 reqired cell lysis step It is ideal for luminometer with automated injectors e Luminometer Generalized Transfection and Assay Protocols The following procedure is designed to transfect the reporter to 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 manufacture s transfection protocol Transfection condition should be optimized according to the cell type and study requirement 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 C
Download Pdf Manuals
Related Search