Data Sheet - BPSBioscience.com
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1. assay below in at least triplicate Add three fold serial dilution of human TGF 1 in 5 ul of assay medium to stimulated wells Add 5 ul of assay medium to unstimulated control wells Add 55 ul of assay medium to cell free control wells for determining background luminescence 5 Incubate at 37 C in a CO incubator overnight 18 hours 6 Perform dual luciferase assay using Dual Glo Luciferase Assay System Add 55 u of Luciferase reagent per well rock at room temperature for 15 minutes and measure firefly luminescence using a luminometer Add 55 1 of Stop amp Glo reagent per well rock at room temperature for 15 minutes and measure Renilla luminescence 7 To obtain the normalized luciferase activity for the SBE reporter subtract background luminescence then calculate the ratio of firefly luminescence from the SBE 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 SBE reporter activity to human TGFB1 The results are shown as fold induction of normalized SBE l2. normalized luciferase activity of the SBE reporter subtract background luminescence then calculate the ratio of firefly luminescence from the SBE reporter to Renilla luminescence from the control Renilla luciferase vector Figure 2 Inhibition of TGF61 induced SBE reporter activity by SB525334 Figure 2a SB525334 completely blocks TGF 1 induced SBE reporter activity Normalized Luciferase Activity Oo TGFB SB52533 4 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 bpsbioscience com Figure 2b Dose response of TGFB1 induced SBE reporter activity to SB525334 The results are shown as percentage of SBE reporter activity The normalized luciferase activity for cells stimulated with TGFB1 in the absence of SB525334 was set at 100 The IC50 of SB525334 is 0 032 uM IC50 0 032 uM 120 Relative Luciferase oO O SB525334 log uM Reference Moustakas A et al 2001 Smad regulation in TGF beta signal transduction J Cell Science 114 Pt 24 4359 69 Related Products Product Cat Size SBE Reporter HEK293 Cell Line 60653 2 vials TGFB1 Active
3. transfections are 1 wl of Reporter component A negative control siRNA 1 pl of Negative Control Reporter component B specific siRNA and 1 wl of Negative Control Reporter component B negative control siRNA Note we recommend setting up at least triplicates for each condition and prepare master mix of transfection cocktail for multiple wells to minimize pipetting errors 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 CO incubator After 24 hours of transfection change medium to fresh medium 48 hours after transfection perform the dual luciferase assay following manufacturer s protocol To study the effect of activators inhibitors on the TGFB pathway treat the cells with test activator inhibitor after 24 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
4. 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 SBE Reporter Kit TGFB SMAD signaling pathway Catalog 60654 Background The transforming growth factor beta TGFB signaling pathway is involved in a diverse range of cell processes such as differentiation cell cycle arrest and immune regulation TGF signaling has been linked to cardiac disease cancer Alzheimer s and other human diseases TGFB proteins bind to receptors on the cell surface initiating a signaling cascade that leads to phosphorylation and activation of SMAD2 and SMAD3 which then form a complex with SMAD4 The SMAD complex then translocates to the nucleus and binds to the SMAD binding element SBE in the nucleus leading to transcription and expression of TGFB SMAD responsive genes Description The SBE Reporter kit is designed for monitoring the activity of TGFB SMAD signaling pathway in the cultured cells The kit contains transfection ready SBE luciferase reporter vector which is a TGFB pathway responsive reporter This reporter contains a firefly luciferase gene under the control of multimerized SBE responsive element located upstream of a minimal promoter The SBE 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 vec
5. 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 Sample protocol to determine the dose response of HEK293 cells transfected with SBE reporter to human TGFB1 Additional materials required for this experiment e Human TGF 1 BPS Bioscience 90900 1 e HEK293 growth medium MEM EBSS with L glutamine Hyclone SH30024 01 10 FBS 1 non essential amino acids 1 mM Na pyruvate 1 Pen Strep e HEK293 assay medium MEM EBSS with L glutamine Hyclone SH30024 01 0 5 FBS 1 non essential amino acids 1 mM 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 a white clear bottom 96 well plate in 100 ul of growth medium Incubate cells at 37 C in a CO incubator overnight 2 Next day transfect 1 ul of SBE luciferase reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols 3 After 24 hours of transfection change media to 50 ul assay medium Incubate cells at 37 C in a CO incubator for 4 5 hours 4 After 29 hours of transfection set up each
6. Protein 90900 1 1 ug TGFB1 Active Protein 90900 2 5 ug TGFB1 Active Protein 90900 10 10 ug TGFB1 Active Protein 90900 3 1000 ug TGFB1 Latent Protein 90901 1 5 ug 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
7. 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 2 Next day transfect 1 ul of SBE luciferase reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols 3 After 24 hours of transfection treat transfected cells with three fold serial dilution of B525334 in 50 ul assay medium Incubate cells at 37 C in a CO incubator for 4 5 hours For wells without SB525334 change to 50 ul assay medium with no treatment 4 After 29 hours of transfection set up each assay below in at least triplicate Add human TGF81 final concentration 20 ng ml in 5 ul of growth medium to stimulated wells cells treated with TGFB1 with or without SB525334 Add 5 ul of assay medium to the unstimulated control wells for determining the basal activity Add 55 ul of growth medium to cell free control wells for determining background luminescence 5 Incubate at 37 C in a CO incubator for overnight 18 hours 6 Perform dual luciferase assay using Dual Glo Luciferase Assay System Add 55 ul of Luciferase reagent per well rock at room temperature for 15 minutes and measure firefly luminescence using a luminometer Add 55 ul of Stop amp Glo reagent per well rock at room temperature for 15 minutes and measure Renilla luminescence 7 To obtain the
8. e 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 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 Incubate the plate at 37 C in a COs incubator 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 pl of Negative Control Reporter component B e 1 ul of Reporter component A experimental vector expressing gene of interest in this experiment the control transfections are 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 wl of Reporter component A specific siRNA in this experiment the control
9. tion 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 lines 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 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 use with luminometers with automated injectors e Luminometer 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 conditions should be optimized according to the cell type and study requirements OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order pleas
10. tor premixed with constitutively expressing Renilla luciferase vector as negative control The non inducible luciferase vector contains a firefly luciferase gene under the 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 TGF signaling pathway activity e Screen activators or inhibitors of TGFB SMAD signaling pathway e Study effects of RNAi or gene overexpression on the activity of TGFB 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 Bi San Diego CA 92121 Tel 1 858 829 3082 IOSCIENCEe Fax 1 858 481 8694 Email info bpsbioscience com Components Component Specification Amount Storage Reporter SBE 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 SUITABLE for transformation and amplica
11. uciferase reporter activity Fold induction was determined by comparing values against the mean value for control cells without TGFR1 treatment The EC50 of TGFB1 is 1 6 ng ml a EC50 1 6 ng ml 10 0 7 5 5 0 Fold Induction 2 5 0 0 4 3 2 1 0 1 2 TGFbeta1 Log ng ml Sample protocol to determine the effect of antagonists of TGFB1 SMAD signaling pathway on TGFB1 induced SBE reporter activity in HEK293 cells Additional materials required for this experiment e B525334 Selleckchem 356559 20 1 inhibitor of TGFB pathway Make stock solution in DMSO e Human TGF 1 BPS Bioscience 90900 1 e HEK293 growth medium MEM EBSS with L glutamine Hyclone SH30024 01 10 FBS 1 non essential amino acids 1 mM Na pyruvate 1 Pen Strep e HEK293 assay medium MEM EBSS with L glutamine Hyclone SH30024 01 0 5 FBS 1 non essential amino acids 1 mM 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 C in a CO incubator overnight 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
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