HEK293-FcγRII cells grown on poly-l -lysine–coated 24-well plates (105 cells/well) were permeabilized as described previously (Arasaki et al., 2012 (link)). Permeabilized cells were incubated with or without 3 µg purified His-DrrA for 1 h at room temperature. Cells were washed, and 100 µl of a PNS fraction from HEK293-FcγRII cells was added with GTP at 0.5 mM final concentration. Cells were incubated for 1 h at room temperature and then washed extensively. To assay tethering, 100 µl lysis buffer (100 mM NaCl, 1 mM MgCl2, 20 mM Hepes-KOH, pH 7.2, 1% Triton X-100, and protease inhibitors) was added to the cells to liberate Luciferase-KDEL from vesicles, and activity was measured using a Luciferase assay kit (New England Biolabs).
Luciferase assay kit
Manufactured by New England Biolabs
The Luciferase assay kit is a laboratory tool used to measure the activity of the luciferase enzyme, which is commonly used as a reporter gene in molecular biology experiments. The kit provides the necessary reagents and protocols to quantify luciferase activity in cell lysates or other biological samples.
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2 protocols using luciferase assay kit
1
DrrA-Mediated Tethering Assay
2
Quantifying TNFR1 Binding Affinity
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Equal aliquots of cells were incubated with conventional TNF (50 µg/ml) for 1 h on ice or left untreated. Cells were then incubated pairwise for another hour with increasing concentrations of GpL-TNC-TNF(32W/86T). Then, cells were washed three times with ice cold PBS to remove unbound GpL-TNC-TNF(32W/86T). Cell pellets were finally suspended in 50 µl RPMI culture medium with 0.5% FBS, transferred to a black 96-well plate and assayed for luciferase activity with the Luciferase Assay Kit from New England Biolabs GmbH using a Lumo laminator (Autobio-Diagnostics). Luciferase activities of GpL-TNC-TNF(32W/86T)-treated cells were considered as total binding and luciferase activities of TNF-pretreated/GpL-TNC-TNF(32W/86T)-treated cells were considered as nonspecific binding. Specific binding values were calculated as the difference between total and non-specific binding values and analyzed by nonlinear regression to a one-site specific binding curve with the GraphPad Prism 5 software to obtain the KD-value of the interaction of TNFR1 with GpL-TNC-TNF(32W/86T).
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