HEK293T cells were seeded in a white-bottom 96-well flat microplate (#655083, CELLSTAR, Greiner Bio-One) at a concentration of 4.0 × 104 cells per well. After 24 h, cells were transfected using PEI MAX (#24765-1, Polysciences) with 100 ng of pSNL-N2-C or pSNL-N2-Muc7 and 100 ng of pSNL-N1-“C interactors.” At 24 h, post-transfection media was removed and Nluc enzymatic activity was measured using a Berthold Centro-XS luminometer by injecting 50 μl of luciferase substrate reagent (#E2820, Promega) per well and counting luminescence for 2 s. Results were expressed as relative light units or as a fold change normalized over the sum of controls, specified herein as normalized luminescence ratio (NLR). For a given protein pair A/B, NLR = (Nluc1-A + Nluc2-B)/[(Nluc1-A + Nluc2) + (Nluc1 + Nluc2-B)]. For the NLR validation experiment, each protein pair was assessed three times or more. The NLR of the protein pair was considered as ‘validated’ if above a threshold value of log(NLR) of 1.55 and above the upper limit of the confidence interval defined for the Muc7/bait pairs.
Luciferase substrate reagent
Manufactured by Promega
Sourced in Germany
The Luciferase substrate reagent is a complex mixture of chemicals that, when combined with luciferase-expressing samples, produces a bioluminescent reaction. The reagent contains the necessary components to facilitate this enzymatic reaction, allowing for the detection and quantification of luciferase activity.
Automatically generated - may contain errors
Lab products found in correlation
Renilla lysis buffer, by Promega (4 mentions)
Centro lb960 luminometer, by Promega (2 mentions)
Cellstar, by Greiner (1 mentions)
Pei max, by Polysciences (1 mentions)
Centro lb 960, by Berthold Technologies (1 mentions)
Td 20 20 luminometer, by Promega (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Fluostar optima, by BMG Labtech (1 mentions)
6 protocols using luciferase substrate reagent
1
Quantification of Protein-Protein Interactions
2
Luminescence-based Transfection Assay
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HEK-293T cells were seeded into white 96-well plates at 3 × 104/well. After 24 h, cells were transfected with linear PEI (polyethylenimine) with 300 ng of a Glc2-PB2-expressing plasmid and 100 ng of a Glc1-UPS-expressing plasmid. At 24 h posttransfection, cells were washed with 100 µl of phosphate-buffered saline and lysed with 40 µl of Renilla lysis buffer (Promega E2820) for 1 h. G. princeps luciferase enzymatic activity was measured with a Berthold Centro LB960 luminometer by injecting 50 µl of luciferase substrate reagent (Promega E2820) per well and measuring luminescence for 10 s. Results were expressed in relative luminescence units.
3
Quantification of Nitric Oxide Synthase Activity
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About one million RAW 264.7 macrophages stably expressing control vector, wild-type PPE2 or ΔNLS-PPE2 were transfected with 2.5 μg inos-luc (Kind gift from Prof. E.D Chan, University of Colorado, Denver) and 0.5 μg pCDNA-LacZ plasmids using Lipofectamine LTX along with plus reagent (3 μg) and at 8 hour post-transfection, cells were stimulated with 3 μg/ml LPS plus 10 ng/ml IFN-γ. Cells harvested at 24 hour post-stimulation were lysed with reporter lysis buffer (Promega, Madison, WI, USA) and protein concentration was estimated with Micro BCATM Protein Assay Kit following the manufacturer’s instructions. About 50 μg protein in 20 μl lysis buffer was mixed with 100 μl of luciferase substrate reagent (Promega). The light emitted was measured using TD 20/20 luminometer (Turner Biosystems, CA, USA). For β-Galactosidase reporter assay, 100 μg of protein was used. The β-Galalactosidase was quantitated using β-Galactosidase reporter assay kit (Roche Applied Science) following the manufacturer’s protocol as described earlier45 (link). The results were expressed as relative luciferase units normalized to β-Galalactosidase activity.
4
Viral Protein-Gaussia Luciferase Fusion Assay
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HEK-293T cells were seeded into white 96-well plates at 3 × 104 cells/well. After 24h, cells were transfected with linear PEI (polyethylenimine) (Polysciences Inc.) with 200 ng of plasmid expressing a viral protein fused to Gluc1 at its N-terminus (PB1, PB1, PA, NP, NS1, NEP) or at its C-terminus (M1) and 100 ng of a Gluc2-ExoRDec-expressing plasmid. At 24 h post-transfection, cells were washed with 100 μl of phosphate buffered saline and lysed with 40 μl of Renilla lysis buffer (Promega E2820) for 30 min at room temperature. Gaussia princeps luciferase enzymatic activity was measured with a Berthold Centro LB960 luminometer by injecting 50 μl of luciferase substrate reagent (Promega E2820) per well and measuring luminescence for 10s. Results were expressed in relative luminescence units.
5
Screening Virus-Host Protein Interactions
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White 96-well plates were seeded with 3.5 × 104 HEK293T cells per well. After 24 h, cells were transfected using JetPRIME with 100 ng of pSPICA-N2-VP3 BTV8 and 100 ng of pSPICA-N1-IFN-β pathway agonists. At 24 h post-transfection, cells were lysed with 40 µL of Renilla lysis buffer (Promega) for 30 min. Gaussia princeps luciferase activity was assessed by injecting 50 µL of luciferase substrate reagent (Promega) on a FLUOstar Optima (BMG Labtech, Ortenberg, Germany). Results were expressed as a fold change normalized over the sum of controls, specified as normalized luminescence ratio (NLR) [26 (link),27 (link)]. For a given protein pair A/B, NLR = (Gluc1-A + Gluc2-B)/[(Gluc1-A + Gluc2) + (Gluc1 + Gluc2-B)]. The NLR of the protein pair was considered as “validated” if above the upper limit of the positivity threshold defined for a random reference set (RSS)/Bait pairs. The RRS contains 14 proteins, supposedly nonbinders proteins, and was kindly provided by Caroline Demeret and Yves Jacob (Institut Pasteur) [26 (link)]. To determine the positivity threshold, we calculated the mean and the standard deviation of the 14 Gluc2-VP3 BTV8 + Gluc1-RRS values and set the positivity threshold for the interactions as mean + (2× standard deviation), corresponding to a 97% confidence interval (CI: 97%).
6
Dual-Luciferase Protein-Protein Interaction Assay
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HEK293T cells were transfected using the reverse transfection method. Transfection mixes containing 100 ng of pSPICA-N2 and 100 ng of pSPICA-N1 plasmids expressing test protein, plus JetPEI (Polyplus transfection) were dispensed in white 96-well plates. HEK293T cells were then seeded on the DNA mixes at a concentration of 4.2 x104 cells per well. At 48 h after transfection, cells were washed with 50 μL of PBS and lysed with 40 μL of Renilla lysis buffer (Promega, E2820) for 30 min with agitation. Gaussia princeps luciferase enzymatic activity was measured using a Berthold Centro LB960 luminometer by injecting 50 μL per well of luciferase substrate reagent (Promega, E2820) and counting luminescence for 10 s. Results are expressed as a fold change normalized over the sum of controls, specified herein as normalized luminescence ratio (NLR) (27 (link)). For a given protein pair A/B, NLR = (Gluc1-A + Gluc2-B)/[(Gluc1-A + Gluc2) +(Gluc1 + Gluc2-B)].
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