Luciferase activities were determined by the Dual Luciferase Stop and Glo Reporter assay system using reagents as described by Dyer et al. (Dyer et al., 2000 (link)), with a slight modification (the homemade Stop and Glo buffer was supplemented with 5% commercial Stop and Glo buffer purchased from Promega). Relative light units were measured on a CentroXS3 LB960 microplate luminometer fitted with two injectors (Berthold Technologies). After removing the media, transfected cells were lysed in 25 μl 1× passive lysis buffer (PLB; Promega), and light emission was measured after injection of 50 μl firefly luciferase substrate followed by the same volume of Renilla luciferase substrate. For phRL mutant series, the Renilla luciferase activity was normalized relative to the activity of an internal control p2luc-based plasmid expressing firefly luciferase from an AUG codon in perfect context (Ivanov et al., 2010c (link)). For p2luc mutant series, the firefly luciferase activity was normalized relative to the activity of a plasmid, pSV40-Renilla, expressing Renilla luciferase (Loughran et al., 2012 (link)). For the antizyme dual luciferase reporters, firefly was normalized to Renilla luciferase made upstream from the same cistron and the values of “wild type” were compared to “in-frame” control to calculate percent frameshifting.
Stop and glo buffer
Manufactured by Promega
Sourced in United States
The Stop and Glo buffer is a solution designed to terminate a reaction and initiate the measurement of luminescence. It serves as a stabilizing agent to preserve the luminescent signal for quantification.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (3 mentions)
Dual luciferase reporter 1000 assay system, by Promega (2 mentions)
Centro xs3 lb960 microplate, by Berthold Technologies (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Larii buffer, by Promega (1 mentions)
Precellys 24, by Bertin Technologies (1 mentions)
Cytation 5, by Agilent Technologies (1 mentions)
4 protocols using stop and glo buffer
1
Dual Luciferase Reporter Assay Protocol
2
Quantifying Luciferase Expression in Ganglia
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Luciferase quantification was performed using a Dual-Luciferase® Reporter Assay System (Promega). Ganglia were disrupted by placing them into a Lysing matrix D-Tube-RNAse/DNAse free Eppendorf (MP Biomedicals) with 200 µL of passive lysis buffer (Precellys 24 Bertin technologies). The tissue lysate was centrifuged for 15 min at 4 °C and 12,000 rpm. Then, 20 µL of supernatant was deposed in a 96-well black plate (Nunc #: 237107) in which 100 µL of LARII buffer (Promega) was added. In a second step, 100 µL of Stop and Glo buffer (Promega) was added to inhibit FLuc expression, and to allow RLuc expression. Luciferase expressions were immediately quantified using a luminometer (FLUOstar Omega BMG LABTECH, France). Promoter strength in any specific tissue was determined by the ratio between the FLuc and RLuc, corresponding to transgene expression normalized by the rate of infection.
3
Transient Dual-LUC Assays in Nicotiana benthamiana
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Transient dual-LUC assays in N. benthamiana were conducted as described previously [37 (link),38 (link)], with minor modifications. After agroinfiltration, plants were placed in a plant growth chamber, and leaf samples were harvested 3 d after infiltration for the dual-LUC assay using Dual-Luciferase Reporter 1000 Assay System (Promega, Madison, WI, USA). In brief, three leaf discs at agroinfiltration areas (5–6 mm in diameter) were excised and ground in liquid nitrogen to fine powder and homogenized in 100 µL Passive Lysis buffer (Promega, Madison, WI, USA). Subsequently, 5 µL of the extract was mixed with 40 µL Luciferase Assay Buffer, and the firefly LUC activity was measured by a cell imaging multimode plate reader (BioTek Cytation 5, Santa Clara, CA, USA). The reaction was stopped by addition of 40 µL Stop and Glo Buffer (Promega, Madison, WI, USA), and the Renilla (REN) LUC activity was measured. The firefly LUC activity was normalized to the REN LUC activity.
4
Transient Dual-Luciferase Assay in N. benthamiana
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Transient dual-luciferase assay in N. benthamiana leaves was performed following the protocols described previously [61 (link),62 (link)] with slight modifications. N. benthamiana plants were put in a plant growth chamber after the agroinfiltration. Leaf discs were collected 3 d post infiltration for the dual-LUC experiment using the Dual-Luciferase Reporter 1000 Assay System (Promega, Madison, WI, USA). Briefly, three leaf discs (5–6 mm in diameter) from the agroinfiltration areas were removed and then ground to fine powder in liquid nitrogen. The powder was subsequently homogenized in 100 µL Passive Lysis buffer (Promega, Madison, WI, USA). Next, 5 µL of the extract was mixed with 40 µL of Luciferase Assay Buffer, and the firefly LUC activity was quantified by a cell imaging multimode plate reader (BioTek Cytation 5, Santa Clara, CA, USA). The reaction was stopped by adding 40 µL Stop and Glo Buffer (Promega, Madison, WI, USA), and the Renilla (REN) LUC activity was quantified. The firefly LUC activity was normalized to the REN LUC activity.
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