Passive reporter lysis buffer

Manufactured by Promega

Sourced in United States

Passive reporter lysis buffer is a solution used to extract and solubilize proteins from cells that have been transfected or transduced with reporter genes. It allows for the efficient lysis of cells and release of reporter proteins, such as luciferase or β-galactosidase, without the need for mechanical disruption.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (4 mentions) Ptkrluc plasmid, by Promega (2 mentions) Prl tk vector, by Promega (1 mentions) Fugene hd, by Promega (1 mentions) Luciferase assay system, by Promega (1 mentions) Cell line nucleofector kit r, by Lonza (1 mentions) Centro lb 960, by Berthold Technologies (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions)

Spelling variants of this product

Reporter lysis buffer (622 citations) Lysis buffer (395 citations) Passive buffer (13 citations) Promega passive lysis buffer (5 citations) Passive Lysis Buffer (Dual-Luciferase Reporter Assay System, (4 citations) Passive Lysis Buffer of the Dual Luciferase Reporter Assay Kit (2 citations)

7 protocols using passive reporter lysis buffer

NF-κB Transcriptional Activity Assay

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The construct pNF-κB-LUC containing five copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene was purchased from Stratagene (La Jolla, CA). Reporter gene transfections and luciferase assays were performed essentially as described^{47 (link)}. Briefly, 5 µg of DNA was mixed with DEAE-dextran (50 µg/ml) in serum-free EBM2, and the resulting mixture was applied onto cells that were 60–80% confluent. 0.125 µg of pTKRLUC plasmid (Promega, Madison, WI) containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter was used to normalize the transfection efficiencies. After 1 h, cells were exposed to 10% DMSO in serum-free EBM2 for 4 min and then washed twice with PBS and allowed to grow to confluency in EBM2–10% FBS. After appropriate treatments, cells were lysed in passive reporter lysis buffer (Promega) and cell extracts were assayed for firefly and Renilla luciferase activities using dual luciferase reporter assay system (Promega).The data were expressed as a ratio of firefly to Renilla luciferase activity.

Leonard A., Grose V., Paton A.W., Paton J.C., Yule D.I., Rahman A, & Fazal F. (2019). Selective Inactivation of Intracellular BiP/GRP78 Attenuates Endothelial Inflammation and Permeability in Acute Lung Injury. Scientific Reports, 9, 2096.

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Analyzing c-Myc Promoter Activity

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HCT116 and SW480 cells were seeded in 24-well plates. At 50–60% confluence, cells were cotransfected with 0.04 μg pGL3-c-Myc-promoter plasmid containing 2.0 kb nucleotide sequence at the upstream region of c-Myc and 0.004 μg pRL-TK vector (Promega) containing renilla luciferase as an internal control using Fugene HD. The cells were then cultured under normoxic or hypoxic conditions for 24 h. 200 μl of passive reporter lysis buffer (Promega) was added to cells. Luciferase activity was analyzed by the dual-luciferase reporter assay system according to the manufacturer's protocols (Promega).

Wang L., Xue M, & Chung D.C. (2016). c-Myc is regulated by HIF-2α in chronic hypoxia and influences sensitivity to 5-FU in colon cancer. Oncotarget, 7(48), 78910-78917.

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NF-κB Transcriptional Activity Assay

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The construct pNF-κB-LUC containing five copies of consensus NF-κB sequences linked to a minimal E1B promoter-luciferase gene was purchased from Stratagene (La Jolla, CA). Reporter gene transfections and luciferase assays were performed essentially as described (2 (link)). Briefly, 2.5 µg of DNA was mixed with 50 µg/ml DEAE-dextran in serum-free EBM2, and the resulting mixture was applied onto cells that were 60–80% confluent. 0.125 µg of pTKRLUC plasmid (Promega, Madison, WI) containing Renilla luciferase gene driven by the constitutively active thymidine kinase promoter was used to normalize the transfection efficiencies. After 1.5 h, cells were exposed to 10% DMSO in serum-free EBM2 for 4 min and then washed twice with PBS and allowed to grow to confluency in EBM2–10% FBS. After appropriate treatments, cells were lysed in passive reporter lysis buffer (Promega) and cell extracts were assayed for firefly and Renilla luciferase activities using dual luciferase reporter assay system (Promega).The data were expressed as a ratio of firefly to Renilla luciferase activity.

Leonard A., Su P.Y., Yule D.I., Rahman A, & Fazal F. (2020). Critical Role of Mortalin/GRP75 in Endothelial Cell Dysfunction Associated with Acute Lung Injury. Shock (Augusta, Ga.), 54(2), 245-255.

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NF-kB and AP-1 Luciferase Assay

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NF-kB and AP-1 luciferase reporter assay was performed as described previously^{27 (link)}. Briefly, Raw 264.7 cells were transfected with plasmid containing NF-kB and AP-1 Luciferase (Genetransfer Vector Core; Iowa City, IA, USA) for 36 h, and the infected with T. gondii for indicated time periods. Infected cells were washed three times in PBS, and cell extracts were prepared by adding 100 μl of 1×Passive Reporter Lysis Buffer (Promega, Madison, WI, USA) Luciferase activity was measured using the Luciferase Assay System (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

Kim J.H., Lee J., Bae S.J., Kim Y., Park B.J., Choi J.W., Kwon J., Cha G.H., Yoo H.J., Jo E.K., Bae Y.S., Lee Y.H, & Yuk J.M. (2017). NADPH oxidase 4 is required for the generation of macrophage migration inhibitory factor and host defense against Toxoplasma gondii infection. Scientific Reports, 7, 6361.

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FGF-23-mediated FGFR/α-Klotho Activation Assay

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For FGF-23-mediated activation of FGFR/α-Klotho complexes, the HEK-293 cells were transiently transfected with α-Klotho membrane or secreted form along with Elk1-Gal4 luciferase reporter system and Renilla luciferase-null as internal control plasmid by electroporation using Cell Line Nucleofector Kit R according to the manufacturer’s protocol (Amaxa, Inc., Gaithersburg, MD, USA). About 48 h after transfection, the transfected cells were treated with 1 nM FGF-23 from different macrophages for 6 h. The cells were then lyzed in 1× passive reporter lysis buffer (Promega), and luciferase activities were measured using an Autolumat Luminometer (Wallac-Berthold, Gaithersberg, MD, USA) and Promega Dual-Luciferase^® Reporter Assay System. Data represent results of at least three separate experiments.

Han X., Li L., Yang J., King G., Xiao Z, & Quarles L.D. (2016). Counter-regulatory paracrine actions of FGF-23 and 1,25(OH)2D in macrophages. FEBS letters, 590(1), 53-67.

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Luciferase Reporter Assay in Drosophila and PC12 Cells

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Luciferase reporter assay was carried out in accordance with our previous report [8 (link)]. For reporter assays in Drosophila melanogaster SL2 cells, pPac-mock, pPac-YY1, and/or pPac-Sp1 vectors were co-transfected with phRG-TK and pGL3-CPR (−204 to +2) reporter vectors into cells by using FuGENE 6 (Roche, Basel, Switzerland). For reporter assays in PC12 cells, pcDNA3-mock or pcDNA3-YY1 vector was co-transfected with phRG-TK and pGL3-CPR (−204 to +2) reporter vectors. At 48 h after transfection, cells were lysed in reporter passive lysis buffer (Promega, WI, USA). Luciferase activity in cell lysates was measured by a luminometer (Turner Designs, San Jose, CA, USA). Relative luciferase units were evaluated after normalization against the phRG activity of each sample. Fold change was expressed as relative luciferase unit with pPac or pcDNA expression plasmid/relative luciferase unit with a mock vector.

Nakayama T., Fukutomi T., Terao Y, & Akagawa K. (2021). Syntaxin 1A Gene Is Negatively Regulated in a Cell/Tissue Specific Manner by YY1 Transcription Factor, Which Binds to the −183 to −137 Promoter Region Together with Gene Silencing Factors Including Histone Deacetylase. Biomolecules, 11(2), 146.

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Protein Quantification and Bioluminescence Assay

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Protein from C2C12 cells were extracted with reporter Passive Lysis buffer (Promega) and protein concentration was quantified by Bradford Standard Assay. Quantification of bioluminescence was performed with a luminometer (Centro LB960, Berthold) using the Dual-Luciferase Reporter Assay (Promega France).

Ainaoui N., Hantelys F., Renaud-Gabardos E., Bunel M., Lopez F., Pujol F., Planes R., Bahraoui E., Pichereaux C., Burlet-Schiltz O., Parini A., Garmy-Susini B, & Prats A.C. (2015). Promoter-Dependent Translation Controlled by p54nrb and hnRNPM during Myoblast Differentiation. PLoS ONE, 10(9), e0136466.

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