Data Sheet - BPSBioscience.com
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1. 55 ng DNA ul Component B luciferase vector Note These vectors 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 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 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 optimize2. Treisman R 1992 The serum response element Trends Biochem Sci 17 10 423 426 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 Related Products Product Name Catalog size EGF human 90201 1 100 ug EGF human 90201 2 500 ug EGF mouse 90200 1 100 ug EGF mouse 90200 2 500 ug ERK1 40055 10 ug ERK2 40299 10 ug MAP3K14 NIK 40090 10 ug MAPKAPK2 MK2 40088 100 ug MAPK10 JNK3 40092 10 ug MEK1 K97R 40075 100 ug MEK1 mouse 40121 10 ug MEK1 human 40123 10 ug MEK1 GST tag 40527 50 ug MEK2 40125 10 ug MEKK2 40122 10 ug MEKK3 40124 10 ug U0126 27012 5 mg 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
3. 1 858 481 8694 Email info bpsbioscience com Figure 1 Serum induced the expression of SRE reporter The results are shown as fold induction of normalized SRE reporter activity Fold induction is determined by comparing values against the mean value for control cells with 0 5 FBS treatment 14 12 o 00 5 FBS m 10 FBS m 20 FBS Fold Induction ez N SRE reporter Negative control reporter Figure 2 EGF induced the expression of SRE reporter The results are shown as fold induction of normalized SRE reporter activity Fold induction are determined by comparing values against the mean value for control cells with 0 5 FBS treatment only 40 35 30 25 20 15 fold induction 10 5 0 EGF ng ml 7 1 10 T 1 10 0 5 FBS 10 FBS 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 3 Dose response of SRE reporter activity to EGF in the presence of 0 5 FBS The results are shown as fold induction of normalized SRE reporter activity Fold induction is determined by comparing values against the mean value for control cells with
4. 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 5 Incubate cells at 37 C in a CO incubator for 6 hours 6 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 7 To obtain the normalized luciferase activity for the SRE reporter subtract the background luminescence then calculate the ratio of firefly luminescence from the SRE reporter to Renilla luminescence from the control Renilla luciferase vector Figure 4 Inhibition of EGF induced SRE reporter activity by ERK pathway inhibitor U0126 The results are shown as percentage of SRE reporter activity The normalized luciferase activity for cells stimulated with EGF in the absence of U0126 is set at 100 The IC50 of U0126 is 0 7 uM Ga EC50 0 71 uM ao so Go O Reporter Activity oO So So N So U0126 Log uM References Wong K K 2009 Recent developments in anti cancer agents targeting the Ras Raf MEK ERK pathway Recent Pat Anticancer Drug Discov 4 1 28 35
5. 6044 Cornerstone Court West Suite E San Diego CA 92121 sAeRiONnerO Tel 1 858 829 3082 Bioscience Fax 1 858 481 8694 Email info bpsbioscience com Data Sheet SRE Reporter Kit MAPK ERK Signaling Pathway Catalog 60511 Background The MAPK ERK signaling pathway is a major participant in the regulation of cell growth and differentiation It can be activated by various extracellular stimuli including mitogens growth factors and cytokines Upon stimulation MEK1 2 phosphorylate and activate ERK1 2 The activated ERK translocates to the nucleus where it phosphorylates and activates transcription factors The TCFs Ternary Complex Factors including Elk1 are among the best characterized transcription factor substrates of ERK When phosphorylated by ERK Elk1 forms a complex with Serum Response Factor SRF and binds to Serum Response Element SRE resulting in the expression of numerous mitogen inducible genes Description The SRE Reporter Kit is designed for monitoring the activity of the ERK signaling pathway and the transcriptional activity of SRF in cultured cells The kit contains a transfection ready SRE luciferase reporter vector which is an ERK pathway responsive reporter This reporter contains the firefly luciferase gene under the control of multimerized SRE responsive elements located upstream of a minimal promoter The SRE reporter is premixed with a constitutively expressing Renilla luciferase vector that serves as a
6. 7 C in a CO incubator for 16 to 18 hours The next day after transfection treat cells with 50 ul of medium containing a high percentage of FBS with or without EGF or medium containing 0 5 FBS with EGF For unstimulated control wells use cells in medium with 0 5 FBS Add 50 ul of growth medium 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 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 SRE reporter subtract the background luminescence then calculate the ratio of firefly luminescence from SRE 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 sLeERnioONnero Tel 1 858 829 3082 Bioscience Fax
7. B negative control expression vector e 1 wl of Reporter component A specific siRNA in this experiment the control 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 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 After 5 to 6 hours of transfection change medium to fresh medium with 0 5 serum Incubate cells at 37 C in a CO incubator overnight 5 The next day induce the SRE reporter with medium containing activators of the ERK pathway such as high percentage of serum or growth factors Incubate cells at 37 C ina CO incubator for 6 hours After 6 hour treatment perform the dual luciferase ass
8. ay following the manufacturer s protocol To study the effect of inhibitors on the ERK pathway after 5 6 hours of transfection treat cells with inhibitors in medium containing 0 5 serum The next day treat cells with activators for 6 hours then perform the luciferase assay 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 serum or EGF on SRE reporter activity 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 overnight at 37 C in a CO incubator The next day transfect 1 ul of SRE reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols After 6 hours of transfection change medium to 50 ul of medium containing 0 5 FBS MEM 0 5 FBS with non essential amino acids Na pyruvate and 1 Pen Strep Incubate cells at 3
9. d 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 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 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 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
10. n internal 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 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 MAPK ERK signaling pathway activity and SRF mediated activity e Screen for activators or inhibitors of the MAPK ERK signaling pathway e Study effects of RNAi or gene overexpression on the activity of the MAPK ERK 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 SRE luciferase reporter vector 500 ul 20 C Component A constitutively expressing Renilla 55 ng DNA ul luciferase vector Negative Control Non inducible luciferase vector 500 ul 20 C Reporter constitutively expressing Renilla
11. out EGF treatment The EC50 of EGF is 0 97 ng ml 14 EC50 0 97 ng ml Fold Induction 2 1 0 1 2 3 EGF Log ng ml Sample protocol to determine the effect of inhibitors of the ERK pathway on SRE reporter activity 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 Incubate cells overnight at 37 C in a CO incubator The next day transfect 1 ul of SRE reporter component A into cells following the procedure in Generalized Transfection and Assay Protocols After 6 hours of transfection treat transfected cells with three fold serial dilution of U0126 MEK inhibitor in 50 ul of medium containing 0 5 FBS For wells without U0126 treat cells with medium containing 0 5 FBS only Incubate cells at 37 C in a CO incubator for 16 to 18 hours The next day after transfection treat the cells with recombinant EGF final concentration 10 ng ml in 50 ul of medium containing 0 5 FBS with U0126 For unstimulated control wells determine the basal activity using cells in medium with 0 5 FBS To determine background luminescence add 50ul of medium to cell free control wells Set up each treatment in at least triplicate 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
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