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1. specific siRNA in this experiment the control transfections are 1 wl 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 setting up each condition in at least triplicate and preparing 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 the complexes to each well containing cells and medium Mix gently by tapping the plate 4 Incubate cells at 37 C in a CO incubator for overnight 5 The next day change medium to assay medium Opti MEM I 0 5 FBS 1 non essential amino acids 1 mM Na pyruvate 1 Pen Strep containing an activator of AP1 such as PMA Incubate cells at 37 C in a CO incubator for 6 to 24 hours After treatment perform the dual luciferase assay following the manufacturer s protocol OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE To place your order please contact us by2. 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 West Suite E San Diego CA 92121 s LeERnioDnro Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Figure 3 Inhibition of PMA induced AP1 reporter activity by JNK inhibitor V The results are shown as normalized AP1 reporter luciferase activity 3 5 gt N an N a wo normalized luciferase activity ba a 0 PMA 10 nM z JNK inhibitor V 10 uM F g References Zhou H et al 2005 Frequency and distribution of AP 1 sites in the human genome DNA Research 11 139 150 Gaillard P et al 2005 Design and synthesis of the first generation of novel potent selective and in vivo active benzothiazol 2 yl acetonitrile inhibitors of the c Jun N terminal kinase J Med Chem 48 14 4596 4607 Related Products Product Name Catalog Size AP1 Reporter HEK 293 cell line 60405 1 vial SRE Reporter Kit MAPK ERK Signaling Pathway 60511 500 reactions MAPK10 JNK3 human 40092 10 ug JNK1 B1 K55M human 40871 100 ug MAP3K14 NIK human 40090 10 ug MAPKAPK2 MK2 human 40088 100 ug JNK1 mouse 40071 10 ug JNK2 human 40113 10 ug JNK3 human 40114 10 ug ERK1 human 40055 10 ug ERK2 human 40299 10 ug ERk2 inactive human 40056 10 ug OUR PRODUCTS ARE FOR RESEARCH USE ONLY NOT FOR DIAGNOSTIC OR THERAPEUTIC USE
3. results are shown as fold induction of normalized AP1 reporter activity Fold induction is determined by comparing values against the mean value for unstimulated control cells The EC50 of PMA is 5 4 nM 17 5 EC50 5 4 nM Fold Induction 2 1 0 1 2 3 PMA Log nM 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 West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Sample protocol to determine the effect of JNK pathway inhibitor on AP1 reporter activity 1 One day before transfection seed 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 overnight at 37 C in a CO incubator The next day transfect 1 ul of AP1 reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols Incubate cells at 37 C in a CO incubator for overnight The next day after transfection prepare 10 mM stock solution of JNK inhibitor V in DMSO Dilute the inhibitor in assay medium to a final concentration of 10 uM Carefully remove the medium from wells and add 45 ul of diluted inhibitor in assay medi
4. 6044 Cornerstone Court West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet AP1 Reporter Kit JNK Signaling Pathway Catalog 60612 Background The stress activated protein kinase c jun N terminal kinase SAPK JNK family of proteins includes mitogen activated protein kinases MAPKs that are activated by stress inflammatory cytokines mitogens oncogenes and inducers of cell differentiation and morphogenesis Upon activation of the SAPK JNK pathway MAP Kinase Kinases phosphorylate and activate JNKs The activated JNKs translocate to the nucleus where they phosphorylate and activate transcription factors such as c Jun The activated c Jun forms homodimers or heterodimers with fos family proteins which bind to the activator protein 1 AP1 response element and induce target gene transcription Description The AP1 Reporter Kit is designed for monitoring the activity of the JNK signaling pathway and the transcriptional activity of AP1 in cultured cells The kit contains a transfection ready AP1 luciferase reporter vector This reporter contains the firefly luciferase gene under the control of multimerized AP1 responsive elements located upstream of a minimal promoter The AP1 reporter is premixed with a constitutively expressing Renilla luciferase vector that serves as an internal control for transfection efficiency The kit also includes a non in
5. 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 West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Sample protocol to determine the effect of PMA on AP1 reporter activity in HEK293 or HeLa cells 1 One day before transfection seed HEK293 or HeLa 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 overnight at 37 C in a CO incubator The next day transfect 1 ul of AP1 reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols Incubate cells at 37 C in a CO incubator overnight The next day after transfection prepare 1 mM stock solution of PMA in DMSO Dilute the PMA stock in assay medium Change cell medium to 50 ul of diluted PMA in assay medium to induce the AP1 reporter For unstimulated control wells treat cells with 50 ul of assay medium without PMA Add 50 ul of assay medium without PMA to cell free control wells to determine the background luminescence Set up each treatment in at least triplicate Incubate cells at 37 C in a CO incubator for 6 hours Perform dual luciferas
6. 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. are designed for transient transfection They are NOT SUITABLE 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 e Transfection reagent for mammalian cell line We use Lipofectamine 2000 Life Technologies 11668027 However other transfection reagents work equally well e Opti MEM Reduced Serum Medium Life Technologies 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 Generalized Transfection and Assay Protocols The following procedure is designed to transfect the reporter into HEK293 or HeLa 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 All amounts and vo
8. ducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as a negative control The non inducible luciferase vector contains the firefly luciferase gene under the control of a minimal promoter without any additional response elements The negative control is critical for determining pathway specific effects and the background luciferase activity Applications e Monitor JNK signaling pathway activity and AP1 mediated activity e Screen for activators or inhibitors of the JNK signaling pathway e Study effects of RNAi or gene overexpression on the activity of the JNK 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 West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Components Component Specification Amount Storage Reporter AP1 luciferase reporter vector 500 ul 20 C Component A constitutively expressing Renilla 60 ng DNA ul luciferase vector Negative Control Non inducible luciferase vector 500 ul 20 C Reporter constitutively expressing Renilla 60 ng DNA ul Component B luciferase vector Note These vectors
9. e 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 the AP1 reporter subtract the background luminescence then calculate the ratio of firefly luminescence from AP1 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 West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Figure 1 PMA induced the expression of AP1 reporter The results are shown as fold induction of normalized AP1 reporter activity Fold induction is determined by comparing values against the mean value for unstimulated control cells 14 z gt 8 ao z 6 o uw 4 2 0 PMA 10 nM AP1 reporter AP1 reporter Negative control in HEK293 in Hela reporter Figure 2 Dose response of AP1 reporter activity to PMA in HEK293 cells The
10. lumes in the following setup are given on a per well basis 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 West Suite E San Diego CA 92121 sAeRiONneroO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com 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 The 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 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 ul of Reporter component A
11. um to the wells Add 45 ul of assay medium without inhibitor to inhibitor control wells or unstimulated control wells Add 45 ul of assay medium without inhibitor 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 1 hour Add 5 ul of diluted PMA in assay medium to wells The final PMA concentration for the cells is 10 nM Add 5 ul of assay medium without PMA to the unstimulated control wells cells without inhibitor and without PMA treatment for determining the basal activity Add 5 ul of assay medium without PMA to cell free control wells Incubate cells at 37 C in a CO incubator for 6 hour 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 the AP1 reporter subtract the background luminescence then calculate the ratio of firefly luminescence from the AP1 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
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