Phrg tk renilla

Manufactured by Promega

The PhRG-TK/Renilla is a dual-reporter gene assay system designed for measuring the activities of two reporter genes simultaneously. It contains the Renilla luciferase reporter gene and the herpes simplex virus thymidine kinase (HSV-TK) reporter gene. The core function of this product is to provide a reliable and efficient tool for researchers to monitor and quantify the expression of two different reporter genes in the same experimental system.

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Lab products found in correlation

Dual luciferase kit, by Promega (2 mentions) Gal4 plasmid pm, by Takara Bio (2 mentions) Dual luciferase assay reagent, by Promega (1 mentions) Glomax 96 microplate luminometer, by Promega (1 mentions) Pvp16 plasmid, by Takara Bio (1 mentions) Lipofectamine, by Thermo Fisher Scientific (1 mentions) Passive lysis buffer (plb), by Promega (1 mentions) Fugene 6, by Promega (1 mentions)

3 protocols using phrg tk renilla

Dual-Luciferase Assay for AP1 Activation

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U2OS.rGR cells were grown in DMEM media supplemented with 10% FBS and 0.1 mg/ml G418 and seeded at 30,000 cells per well in a volume of 300 μl per well in 24-well plates as previously described [29 (link)] with the following modifications. One day after seeding in 24-well plates, cells in FBS-free DMEM were transfected with reporter (AP1Luc), 10 ng of phRG-TK Renilla (Promega) as an internal control, and the indicated amounts of plasmids for various factors in OPTIMEM plus XTREME Gene HP (Roche; 0.8 μl/well). Four hours after transfection, cells were refed with DMEM/10% FBS. The next day, cells were treated with PMA (10–25 ng/ml) and various dilutions of dexamethasone (Dex) and chemical. Sixteen hours later, the cells were lysed in lysis buffer and assayed for reporter gene activity using dual luciferase assay reagents according to the manufacturer’s instructions (Promega, Madison, WI). Luciferase activity was measured by a GloMax® 96 Microplate Luminometer (Promega, Madison, WI). The data were normalized to Renilla TK luciferase activity and expressed as a percentage of the maximal response with Dex before being plotted ± S.D. unless otherwise noted.

Chow C.C., Finn K.K., Storchan G.B., Lu X., Sheng X, & Simons SS J.r. (2015). Kinetically-Defined Component Actions in Gene Repression. PLoS Computational Biology, 11(3), e1004122.

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Constructing and Using Gal4 and VP16 Fusion Constructs for Transcriptional Regulation Studies

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To construct the Gal4 DBD-NINJA plasmid, the Arabidopsis NINJA full-length coding sequence was polymerase chain reaction (PCR)–amplified, digested, and ligated into the Gal4 plasmid pM (Clontech). To generate VP16 fusion constructs, full-length and truncated coding sequences of rice TPR2, TPR1, and TPL were PCR-amplified from pLexA-N fusion plasmids (13 (link)), digested, and inserted into the pVP16 plasmid (Clontech). The reporter plasmid pG5-Luc contains a luciferase gene under the control of a Gal4 upstream activating sequence element. Gal4 fusion constructs (30 ng) were cotransfected with VP16 fusion constructs (30 ng), together with 100 ng of pG5-Luc and 1 to 3 ng of phRG-TK/Renilla (Promega), into AD293 cells in 24-well plates by Lipofectamine (Invitrogen) method according to the manufacturer’s manual. Cells were harvested 17 hours after transfection with 1× Passive Lysis Buffer (Promega). Luciferase/Renilla activities were measured with the Dual-Luciferase Kit (Promega), and data were plotted as firefly luciferase activity relative to Renilla luciferase activity.

Ke J., Ma H., Gu X., Thelen A., Brunzelle J.S., Li J., Xu H.E, & Melcher K. (2015). Structural basis for recognition of diverse transcriptional repressors by the TOPLESS family of corepressors. Science Advances, 1(6), e1500107.

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Gal4 Transactivation Assay for Protein Interactions

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To construct the Gal4 DBD-D53 plasmid, full-length, codon-optimized D53 coding sequence was synthesized by GeneWiz with flanking restriction sites. The restriction fragment was cloned into the Gal4 plasmid pM (Clontech). The full-length TPL/TPR1/TPR2-VP16 activation domain constructs were described previously (25 (link)). Gal4 fusion constructs (25 ng) were cotransfected with VP16 fusion constructs (25 ng), together with 100 ng of pG5-Luc reporter and 5 ng of phRG-TK/Renilla (Promega) control into AD293 cells using FuGENE 6 (Promega) according to the manufacturer’s instructions. Cells were harvested 24 hours after transfection and lysed in 1× passive lysis buffer (Promega). Luciferase/Renilla activities were measured with the Dual Luciferase Kit (Promega), and data were plotted as relative activities (Luciferase activity:Renilla activity).

Ma H., Duan J., Ke J., He Y., Gu X., Xu T.H., Yu H., Wang Y., Brunzelle J.S., Jiang Y., Rothbart S.B., Xu H.E., Li J, & Melcher K. (2017). A D53 repression motif induces oligomerization of TOPLESS corepressors and promotes assembly of a corepressor-nucleosome complex. Science Advances, 3(6), e1601217.

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