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Luciferase reporter constructs

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Luciferase reporter constructs are genetic tools that contain the luciferase gene, which encodes an enzyme that catalyzes a bioluminescent reaction. These constructs can be used to monitor gene expression and cellular activities in various experimental systems.

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Lab products found in correlation

Renilla luciferase construct, by Promega (2 mentions) Dual luciferase reporter system, by Promega (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Dual luciferase reporter assay system kit, by Promega (1 mentions) Lipofectamine 2000 reagent, by Promega (1 mentions) Dual luciferase assay, by Promega (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Reporter lysis buffer, by Promega (1 mentions) Tubulin, by Cell Signaling Technology (1 mentions) Sb203580, by Promega (1 mentions) Ly294002, by Promega (1 mentions) Pn3 sp1 fl, by Addgene (1 mentions) Pn3 sp3 fl, by Addgene (1 mentions) P erk, by Merck Group (1 mentions) Mg 132, by Promega (1 mentions) P thr, by Merck Group (1 mentions) P tyr, by Merck Group (1 mentions) Actinomycin d, by Merck Group (1 mentions) U0126, by Promega (1 mentions)

4 protocols using luciferase reporter constructs

1

Luciferase Assay for microRNA Activity

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A dual-luciferase reporter system developed by Promega (E1960, Madison, WI, USA) was used to perform luciferase activity assay. Briefly, U343 cells (ATCC, Manassas, VA, USA) were cultured on 12-well tissue culture plates at a density of 2 × 105 cells per well. Cells were co-transfected with the luciferase reporter constructs (Madison, WI, USA), corresponding miRNA mimics, and Renilla luciferase construct (Promega) for 5 h. After 24 h culture, the transfected cells were lysed by 150 μl of passive lysis buffer; 30 μl of lysates were mixed with 50 μl of LAR II, and then firefly luciferase activity was measured by a luminometer. For the internal control, 50 μl of Stop & Glo reagent (Madison, WI, USA) was added to the sample.
Du W.W., Yang W., Fang L., Xuan J., Li H., Khorshidi A., Gupta S., Li X, & Yang B.B. (2014). miR-17 extends mouse lifespan by inhibiting senescence signaling mediated by MKP7. Cell Death & Disease, 5(7), e1355-.
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2

Validation of miR-140-3p Binding Sites

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Fragments of the C-Myb or BCL-2 3′-UTR containing the putative wild-type or mutant miR-140-3p binding sites were amplified and cloned into luciferase reporter constructs (Promega Biotech, USA) according to the manufacturer's instruction. The constructs were cotransfected into HEK 293 cells with empty vehicle, miR-140-3p mimic (50 nM), mimic oligocontrol (50 nM), miR-140-3p inhibitor (100 nM), or inhibitor oligo-control (100 nM) using Lipofectamine 2000 (Invitrogen, USA). Forty-eight hours after transfection, the firefly or Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega Biotech, USA). Renilla luciferase activity was normalized by firefly luciferase activity.
Zhu Z.R., He Q., Wu W.B., Chang G.Q., Yao C., Zhao Y., Wang M, & Wang S.M. (2018). MiR-140-3p is Involved in In-Stent Restenosis by Targeting C-Myb and BCL-2 in Peripheral Artery Disease. Journal of Atherosclerosis and Thrombosis, 25(11), 1168-1181.
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3

Transient Transfection in JEG-3 Cells

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Transient transfections in JEG-3 cells were performed by using Lipofectamine 2000 reagent (Invitrogen) as per the manufacturer’s instructions. In each well of 24-well plates, the cells were transfected with 2 μl Lipofectamine 2000 reagent, 2 μg luciferase reporter constructs, and 0.02 μg Renilla luciferase constructs (Promega) for 8 hrs in the absence of serum, then, the cells were cultured in the media containing either CM or SM for 48 hrs. In some experiments, the cells were infected with the viruses for 24 hrs, and then transfected with the luciferase reporters for 8 hrs, and finally cultured in the media containing either CM or SM for 48 hrs. After transfection and treatment, the cellular lysates were prepared in reporter lysis buffer (Promega), and the supernatants were used for dual-luciferase assay according to the manufacturer’s instructions (Promega). The firefly luciferase activities were normalized to Renilla luciferase levels.
Tang C., Pan Y., Luo H., Xiong W., Zhu H., Ruan H., Wang J., Zou C., Tang L., Ikuchi T., Long F, & Wu X. (2015). Hedgehog signaling stimulates the conversion of cholesterol to steroids. Cellular signalling, 27(3), 487-497.
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4

Detailed Sodium Butyrate Experimental Protocol

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Sodium butyrate was purchased from Sigma. Pharmacological inhibitors were from Sigma [Actinomycin-D, Cycloheximide, Mithramycin A, H-7, Staurosporin, Rp-CAMP, Sodium orthovanadate (vanadate), Bisindolylmaleimide-I, Go6976, Rottlerin and Okadaic acid], Calbiochem (H-89, U0126, SB203580, JNK-II and MG-132) and Promega (LY294002). Antibodies were purchased from Abcam (TLR5 and Sp1), Santacruz Biotechnology [Sp1 (sc-14027), Sp3 (sc-644 and sc-13018), PKC-δ (sc-213), p300 (sc-584X), p-Ser(4A3) (sc-81516), egr-1 (sc-189), c-myc (sc-789), TLR5 (sc-10742) and tubulin (sc-5286)], Cell Signaling (H3, H4, p-PKC-δ, ERK, p-ERK, p-Thr, p-Tyr, Acetyl-Lys), Sigma (HDAC1) and Upstate (Acetyl-H3 and Acetyl-H4). All others chemicals were purchased from Sigma. Luciferase reporter constructs were bought from Promega. egr-1 overexpression construct and TLR5 promoter deletion reporter constructs were generated in laboratory. pN3-Sp3FL (Addgene plasmid # 24541) and pN3-Sp1FL (Addgene plasmid # 24543) were gifts from Guntram Suske. Primers used for the study are listed in Supplementary Table S1.
Thakur B.K., Dasgupta N., Ta A, & Das S. (2016). Physiological TLR5 expression in the intestine is regulated by differential DNA binding of Sp1/Sp3 through simultaneous Sp1 dephosphorylation and Sp3 phosphorylation by two different PKC isoforms. Nucleic Acids Research, 44(12), 5658-5672.
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