Luciferase activity assay kit

Manufactured by Beyotime

Sourced in China

The Luciferase activity assay kit is a tool designed to measure the activity of the luciferase enzyme. Luciferase is a widely used reporter protein that emits light upon catalyzing a bioluminescent reaction. This kit provides the necessary reagents and protocols to quantify the activity of luciferase in various experimental systems.

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Lab products found in correlation

Pmirglo control vector, by Promega (1 mentions) Mirna mimics or inhibitor, by GenePharma (1 mentions) Microplate reader, by Molecular Devices (1 mentions) Multifunctional enzyme marker, by Agilent Technologies (1 mentions) Psicheck 2 vector, by Promega (1 mentions) Glomax 20 20 luminometer, by Promega (1 mentions)

4 protocols using luciferase activity assay kit

3'-UTR LIP mRNA Luciferase Assay

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The 3′-UTR LIP mRNA wild-type and mutated sequences (GenBank accession No. MT176433) were cloned into the pmirGLO control vector (Promega, Madison, WI, USA) to obtain the pmirGLO-lip-3′-UTR-WT/MUT plasmids, respectively. The same sequences were cloned into the pEGFP-C1 vector to obtain the pEGFP-lip-3′-UTR-WT/MUT plasmids, respectively.
HEK293 cells were seeded on 48-well plates overnight before transfection and then transfected with 25 ng of dual-luciferase reporter WT/MUT vector and 50 nM of miRNA mimics or inhibitor (GenePharma, Suzhou, CHN). Dual-luciferase reporter assays were performed 24 h after transfection using the luciferase activity assay kit, following the manufacturer’s instructions (Beyotime, Beijing, CHN). Firefly luciferase activity was normalized to Renilla luciferase activity. Relative light unit (RLU) data were measured using a microplate reader (Molecular Devices, San Jose, CA, USA) in luminescence detection mode.

Ma L., Gou M., Du Z., Zhu T., Li J., Li Q.W, & Pang Y. (2021). MicroRNA expression profile in Lampetra morii upon Vibrio anguillarum infection and miR-4561 characterization targeting lip. Communications Biology, 4, 995.

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Verification of miR-195 and HOXA10 Interaction

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Genes targeted by miR-195 and LINC00355 were predicted by the biological prediction website http://www.microrna.org/, and whether HOXA10 was a direct target gene of miR-195 was verified using the dual-luciferase reporter gene assay. The target gene fragment was introduced into pMIR-reporter using endonuclease locus SpeI and Hind III. Complementary mutation sites of seed sequence were designed on the miR-195 WT. After restriction endonuclease digestion, the target fragment was inserted into the pMIR-reporter with T4 DNA ligase. The correctly sequenced luciferase reporter plasmids WT and mutant (Mut) were respectively co-transfected with LINC00355 into HEK293T (CRL-1415, Shanghai Xin Yu Biotech, Shanghai, China). After 48 h of transfection, the cells were collected, lysed, and centrifuged for 3–5 min with the supernatant collected. Ratios of firefly/Renilla luciferase were measured using the luciferase activity assay kit (RG005, Beyotime Institute of Biotechnology, Shanghai, China). This method is also applicable to verification of the relation between HOXA10 and miR-195.

Lu S., Sun Z., Tang L, & Chen L. (2019). LINC00355 Promotes Tumor Progression in HNSCC by Hindering MicroRNA-195-Mediated Suppression of HOXA10 Expression. Molecular Therapy. Nucleic Acids, 19, 61-71.

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Transfection and Luciferase Assay in 293 T Cells

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293 T cells were seeded in a 24-well plate with 500 µl complete medium and co-transfected with reporter gene plasmids and transcription factor plasmids. Cell lysis buffer was added 48 h after transfection, and luciferase activity was measured by luciferase activity assay Kit (Beyotime Biotechnology). The Renilla luciferase plasmid was used as an internal control. Chemiluminescence was measured using a multifunctional enzyme marker (Biotek).

Li Z., Zhao Y., Pan Z., Cai B., Zhang C, & Jiao J. (2024). LncRNA-LncDACH1 mediated phenotypic switching of smooth muscle cells during neointimal hyperplasia in male arteriovenous fistulas. Nature Communications, 15, 3743.

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Validating miR-214 Binding to KPNA3 3'UTR

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Karyopherin subunit alpha 3 3′UTR gene fragment containing putative binding site of miR-214 was introduced into psiCHECK2 vector (Promega). The MUT form, in which the binding site was mutated, was designed based on the seed sequence KPNA3 WT. The MUT gene fragment was also inserted into the psiCHECK2 reporter plasmid. The WT and MUT luciferase reporter plasmids were respectively co-transfected with miR-214 mimic into the HEK-293T cells (CRL-1415, Shanghai Xinyu Biological Technology Co., Ltd., Shanghai, China). After 48 h, the cells were collected and lysed, and the luciferase activity was detected on Glomax 20/20 luminometer (Promega) using a luciferase activity assay kit (RG005, Beyotime). The same method was used to detect the relationship between HOTTIP and miR-214.

Chen X., Liu Y., Zhang Q., Liu B., Cheng Y., Zhang Y., Sun Y., Liu J, & Gen H. (2021). Exosomal Long Non-coding RNA HOTTIP Increases Resistance of Colorectal Cancer Cells to Mitomycin via Impairing MiR-214-Mediated Degradation of KPNA3. Frontiers in Cell and Developmental Biology, 8, 582723.

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