Reporter lysis buffer

Manufactured by Promega

Sourced in United States, United Kingdom, Germany, France, Italy

The Reporter Lysis Buffer is a solution designed to lyse cells and release reporter proteins, such as luciferase, for analysis. It is a core component in a variety of reporter gene assays.

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Lab products found in correlation

Luciferase assay system, by Promega (84 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (60 mentions) Luciferase assay reagent, by Promega (53 mentions) Luciferase assay kit, by Promega (28 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (26 mentions) Dual luciferase reporter assay system, by Promega (23 mentions) β galactosidase enzyme assay system, by Promega (16 mentions) Luciferase substrate, by Promega (13 mentions) Dual glo luciferase assay system, by Promega (12 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (12 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (12 mentions) Glomax luminometer, by Promega (11 mentions) Glomax 20 20 luminometer, by Promega (11 mentions) Bca protein assay kit, by Thermo Fisher Scientific (10 mentions) Dual luciferase assay kit, by Promega (9 mentions) Pgl3 basic vector, by Promega (8 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions) Glomax multi detection system, by Promega (7 mentions) Lipofectamine, by Thermo Fisher Scientific (7 mentions) Prl tk, by Promega (7 mentions)

622 protocols using reporter lysis buffer

EGCG Pretreatment and E7 Transfection in NHEK Cells

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NHEK cells were seeded (1 × 10⁵ cells/well) into 6 well plates and were pretreated with 50 μM of EGCG for 2 h prior to transfection. Untreated cells were incubated with the same amount of sterile distilled water used as EGCG solvent for the same treatment time. Subsequently, the cells were co-transfected with empty vector or E7 with pCMV-βgal-R1 vector using TransIT-X2^® transfectant (Mirus) for 24 h according to the manufacturer’s instructions. After transfection, cells were lysed with 5X reporter lysis buffer (Promega, Madison, WI, USA) and then used for assays. β-galactosidase assay was used to β-galactosidase enzyme assay system with reporter lysis buffer (E2000; Promega) according to the manufacturer’s instructions. The absorbance was measured at a wavelength of 420 nm using VersaMax microplate reader (Molecular Devices).

Song J.Y., Han J.H., Song Y., Lee J.H., Choi S.Y, & Park Y.M. (2021). Epigallocatechin-3-gallate Can Prevent Type 2 Human Papillomavirus E7 from Suppressing Interferon-Stimulated Genes. International Journal of Molecular Sciences, 22(5), 2418.

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Modulation of Notch Signaling in HEK293 Cells

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HEK293 cells in a 24-well plate were cotransfected with 100 ng per well Hey1 luciferase reporter gene, 300 ng per well pCMV-NICD (generous gift from Dr. Eileen M. Redmond, Department of Surgery, University of Rochester School of Medicine and Dentistry) and 100 ng per well CMV-β-galactosidase (β-gal) using FuGENE® HD (Promega). After 12-h transfection, cells were treated with 30-μM TAT-Sc or TAT-ANK for another 12 h. A luciferase assay system with reporter lysis buffer (Promega, E4030) was used to detect the luciferase activity. β-Gal activity was measured by β-gal enzyme assay system with reporter lysis buffer (Promega, E2000).

Zhu G., Lin Y., Ge T., Singh S., Liu H., Fan L., Wang S., Rhen J., Jiang D., Lyu Y., Yin Y., Li X., Benoit D.S., Li W., Xu Y, & Pang J. (2022). A novel peptide inhibitor of Dll4-Notch1 signalling and its pro-angiogenic functions. British journal of pharmacology, 179(8), 1716-1731.

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Luciferase Assay for ROB and DOC Treatment

Cited 1 time

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PC-3/N cells³⁵ (link) were treated with ROB and/or DOC for 24 h, and then the cells were harvested in 1× reporter lysis buffer (Promega, Madison, WI, USA). After centrifugation, 10 µl aliquots of the supernatants were mixed with 10 µl of luciferase substrate (Promega) and measured for the luciferase activity by using a Luminometer LuMate™ (Awareness Technology, Palm City, FL, USA). The luciferase activity was normalised against known protein concentrations and expressed as the percentage of luciferase activity in the control cells. The protein level was determined by Bio-Rad protein assay kits (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions.

Wang X., Xuetao X., Wu M., Wu P., Sheng Z., Liu W., Ma Y.Y., Zhao D.G., Zhang K., Li D., Zheng X, & Goodin S. (2022). Inhibitory effect of roburic acid in combination with docetaxel on human prostate cancer cells. Journal of Enzyme Inhibition and Medicinal Chemistry, 37(1), 542-553.

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Coculture Assay for PAI-1 Expression

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MLEC stably transfected with the PAI-1 luciferase promoter construct were used. They were cocultured with MCs in MEM with 10% FBS, plated at 5000 and 25,000 cells/well, respectively, on a 12-well plate (1:5 ratio MLEC:MC). The next day, cells were serum deprived for 18 h followed by treatment with HG and inhibitors. At collection, cells were lysed in 1× Reporter Lysis Buffer (Promega) and stored at −80 °C overnight before analysis of PAI-1 luciferase activity.

Trink J., Li R., Squire E., O’Neil K., Zheng P., Gao B, & Krepinsky J.C. (2022). Integrin β1/Cell Surface GRP78 Complex Regulates TGFβ1 and Its Profibrotic Effects in Response to High Glucose. Biomedicines, 10(9), 2247.

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Dual Luciferase Reporter Assay Protocol

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Dual Luciferase Report Assay were performed according to the manufacturer's protocol (Dual-Glo^® luciferase assay system, #E2940, Promega Corporation, Madison, WI, USA). In brief, 0.6 µg luciferase reporter and were cotransfected along with 0.3 µg pCMV-lacZ (Promega Corporation) plasmid into the 786-O cells (SCR and shKMT5A). At 48 h following transfection, these cells were harvested and lysed in 1× reporter lysis buffer (Promega Corporation). Luciferase activities were determined with luciferase assay system (Promega Corporation) according to the manufacturer's protocol. The method of normalization was the Firefly comparison with Renilla luciferase activity.

Lin Z.Z., Ming D.S., Chen Y.B., Zhang J.M., Chen H.H., Jiang J.J, & Zhang Z.S. (2019). KMT5A promotes metastasis of clear cell renal cell carcinoma through reducing cadherin-1 expression. Oncology Letters, 17(6), 4907-4913.

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Myogenin Promoter Regulation by AVP and LY294002

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L6 cells were plated in 6-well 35-mm culture dishes at a density of 2.5 × 10⁵ cells/well. Twenty-four hours later, cells were transfected with 1.5 µg of expression vectors driven by the myogenin promoter fragments: pMyo84-luc, pMyo84(mMEF2)-luc and pMyo84(-E1)-luc using the Lipofectamine reagent (Thermo Scientific, Rockford, IL, USA) following the manufacturer’s instructions. The plasmid encoding β-galactosidase under the control of the cytomegalovirus (CMV) promoter, CMV-βgal, was included to monitor transfection efficiency and for normalization of reactions. At 24 h after the start of transfection, the transfection mixture was removed and replaced with DMEM + 1% bovine serum albumin and the cells were treated with or without 0.1 μM AVP, 20 μM LY294002 and AVP + LY294002 for 48 h. Cells were then washed twice with PBS and scraped in 1× reporter lysis buffer (Promega, Milan, Italy). Luciferase activity was determined with a luciferase assay kit (Promega, Milan, Italy).

Sorrentino S., Barbiera A., Proietti G., Sica G., Adamo S, & Scicchitano B.M. (2019). Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation. International Journal of Molecular Sciences, 20(17), 4188.

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Luciferase Assay for DHA-Induced Transcription

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MCF-10A cells were plated in a 6-well plate and co-transfected with 1.5 μg of the luciferase reporter gene fusion construct (pTi-luciferase), wild-type ARE, and 0.5 μg of pCMV-β-galactosidase control vector with WelFect-M^™ Gold transfection reagent (WelGENE Inc., Gyeongsan, Korea) according to the manufacturer’s instructions. After 24-h transfection, the cells were treated with DHA for additional 9 h, and cell lysis was carried out with the 1× reporter lysis buffer (Promega, Madison, WI, USA). After mixing the cell extract with a luciferase substrate (Promega, Madison, WI, USA), the luciferase activity was measured by the luminometer (AntoLumat LB 953, EG&G Berthold, Bad Widbad, Germany). The β-galactosidase assay was done according to the supplier’s instructions (Promega, Madison, WI, USA) for normalizing the luciferase activity.

Bang H.Y., Park S.A., Saeidi S., Na H.K, & Surh Y.J. (2017). Docosahexaenoic Acid Induces Expression of Heme Oxygenase-1 and NAD(P)H:quinone Oxidoreductase through Activation of Nrf2 in Human Mammary Epithelial Cells. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(6), 969.

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Luciferase Reporter Assay with KLF10

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CACC-TK-luc plasmid contains three tandem copies of CACCC sites upstream to a luciferase reporter in pGL3-basic as previously described.^{12 (link), 44 (link)} Empty vector pCMV-SPORT6 and human KLF10 expressing plasmid pCMV-SPORT6-KLF10 are from Open Biosystems (Huntsville, AL). HeLa cells from ATCC were cultured in DMEM medium with 10% Fetal Bovine Serum, 1% antibiotics. Cells were transfected by using Fugene® 6 transfection reagent from Roche. In brief, cells were plated (40 000/well) in triplicate in a 12-well plate. After growing to 70–80% confluency, cells were transfected with 500 ng of CACC-TK-luc reporter construct, 200 ng β-galactosidase expression plasmids, and 100 ng pcDNA3.1 empty vector or pcDNA3.1-KLF10. After 24 h incubation, cells were treated with 100 μM or indicated concentration of small molecule compounds for 24 h. Treated cells were collected in 200 μl 1 × Reporter Lysis Buffer (Promega). The activity of luciferase were measured and normalized to total protein read for the same lysate performed in triplicate replicates.

Khedkar S.A., Sun X., Rigby A.C, & Feinberg M.W. (2015). Discovery of Small Molecule Inhibitors to Krüppel-Like Factor 10 (KLF10): Implications for Modulation of T Regulatory Cell Differentiation. Journal of medicinal chemistry, 58(3), 1466-1478.

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Investigating H. pylori-induced TRPC6 regulation

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TRPC6 promoter was amplified from human cDNA, and cloned into the KpnI and BglII sites of a PGL3-basic vector (Promega, USA). Similar plasmids containing mutations in the putative TCF/LEF-binding sites, named Mut, were constructed. AGS and MKN45 cells were co-transfected with the constructed luciferase vectors. Then, H. pylori was added to the cells. After 24 hrs, the cells were harvested in 1×Reporter Lysis Buffer (Catalog no.: E1960, Promega), and luciferase activities were measured using the Dual-Luciferase Reporter Assay System Kit (Promega, WI, USA). The luciferase activity for each lysate was normalized to the renilla luciferase activity.

Song Y., Liu G., Liu S., Chen R., Wang N., Liu Z., Zhang X., Xiao Z, & Liu L. (2019). Helicobacter pylori upregulates TRPC6 via Wnt/β-catenin signaling to promote gastric cancer migration and invasion. OncoTargets and therapy, 12, 5269-5279.

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Evaluating E. daniellii Extract Effects

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HEK293-TOP cells were seeded into 96-well plates at a density of 2.5 × 10⁴ cells/well and were incubated in a medium containing 10% FBS for 24 h. The cells were incubated for 24 h with or without 1, 5, 10, 25, 50 μg/mL of E. daniellii extract or 1, 5, 10, 25, 50 μM of each of its components (Sigma-Aldrich). The total cell lysates were extracted with 25 μL of 1× reporter lysis buffer (Promega, Madison, WI, USA) per well, and luciferase activity was measured using a microplate luminometer (BMG Labtech, Offenburg, Germany).

Yoon M., Seo S.H., Choi S., Han G, & Choi K.Y. (2022). Euodia daniellii Hemsl. Extract and Its Active Component Hesperidin Accelerate Cutaneous Wound Healing via Activation of Wnt/β-Catenin Signaling Pathway. Molecules, 27(20), 7134.

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