Rluc-ATP4A was expressed in vitro using the TnT SP6 Quick Coupled Transcription/Translation kit (Promega), based on transcription by the SP6 phage RNA polymerase and translation by a rabbit reticulocyte lysate cell-free expression system. Nluc-ATP4B was expressed in eukaryotic cells, using the Expi293 expression system (Thermo Fisher Scientific, Waltham, MA, USA). In the Expi293 expression system, recombinant protein expression is achieved by high efficiency transfection of Expi293F, a derivative of HEK293 cells, adapted to growth in suspension in a defined composition, serum free medium. After 48 h of growth with agitation, transfected Expi293F cells were pelleted and lysed with passive lysis buffer (Thermo Fisher Scientific). Expression of recombinant antigens was assessed by quantification of luciferase activity in the lysates after the addition of Renilla luciferase assay system substrate or NanoGlow substrate (Promega), reconstituted according to the manufacturer instructions, for ATP4A and ATP4B, as appropriate. Luciferase activity was measured using a Berthold Centro xS960 luminometer (Berthold, Germany) and expressed in light units (LU) emitted over a time interval of 2 s. Recombinant antigen preparations were aliquoted and stored frozen at −80 °C.
Renilla luciferase assay system substrate
Manufactured by Promega
The Renilla luciferase assay system substrate is a reagent used to measure the activity of Renilla luciferase, a bioluminescent reporter enzyme. The substrate is added to samples containing Renilla luciferase, and the resulting luminescence is proportional to the enzyme's activity. This assay is commonly used in various research applications, such as gene expression studies and high-throughput screening.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Thermo Fisher Scientific (1 mentions)
Nanoglow substrate, by Promega (1 mentions)
Centro xs960 luminometer, by Berthold Technologies (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Glomax 96 luminometer, by Promega (1 mentions)
Bright glo luciferase assay system substrate, by Promega (1 mentions)
Renilla luciferase assay system lysis buffer, by Promega (1 mentions)
Prism 7, by GraphPad (1 mentions)
Transit 2020 transfection reagent, by Mirus Bio (1 mentions)
4 protocols using renilla luciferase assay system substrate
1
In Vitro and Eukaryotic Expression of ATP4A and ATP4B
2
Detecting EBOV VP40 Antibodies
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The C-terminal domain of Mayinga EBOV Zaire viral protein 40 (VP40; bp 583–981) was cloned into the pRen2 plasmid and transfected into Cos-1 cells generating Renilla luciferase antigen fusion proteins. Cell lysates were harvested and used in immunoprecipitation assays with Protein A/G conjugated agarose beads as described by Burbelo et al [15 (link)]. In the final step, the beads were washed 4 times with buffer A and 1 time with phosphate buffered saline using a vacuum manifold before measuring luciferase activities using the Renilla luciferase assay system substrate (Promega). VP40 positivity was determined if the relative luciferase signal postimmunoprecipitation was at least 3 standard deviations greater than the background signal, as determined from an average of 8 previously identified negative serum samples. Positive controls were obtained from human convalescent patient sera collected during the 2014 Boende, DRC, EBOV outbreak [16 (link)]. Data are presented as relative light units (RLU) of patient over RLU of negative controls; values greater than 1, are considered positive.
3
Measuring Viral Genome Packaging Efficiency
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Cells were washed with PBS and lysed with 100 µL (24-well plates) or 500 µL (six-well plates) Renilla Luciferase Assay System Lysis Buffer (Promega). To measure renilla luciferase activity, cell lysates (20 µL) were mixed with 100 µL Renilla Luciferase Assay System Substrate (Promega). To measure firefly luciferase activity, cell lysates (2 µL) were mixed with 18 µL Renilla Luciferase Assay System Lysis Buffer and 50 µL Bright-Glo Luciferase Assay System Substrate (Promega). The luminescence level was measured, and the relative light units (RLUs) of luciferase were determined using a GloMax 96 luminometer (Promega). To analyze the efficiency of viral genome packaging, the RLUs acquired from cells infected with VLPs were divided by the RLUs acquired from cells transfected with corresponding minigenome plasmids. Bar graphs were drawn using GraphPad Prism 7 software (GraphPad Software, Inc.) and statistically analyzed using an unpaired two-tailed t-test.
4
Expression of EBOV VP40 Luciferase Fusion Proteins
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For expression of Renilla luciferase antigen fusion proteins, we cloned the C-terminal domain of EBOV VP40 (bp positons 583–981) into the pRen2 plasmid and transfected it into Cos-1 cells by using a 10-cm culture dish, 10 μg plasmid DNA, and the TransIT 2020 Transfection Reagent (Mirusbio, https://www.mirusbio.com ). Cell lysates containing the fusion proteins were harvested at 48-h posttransfection. The LIPS assay was performed as described (12 (link)). We measured luciferase activities by using the Renilla Luciferase Assay System Substrate (Promega).
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