For transient reporter activity assays, protoplasts derived from an Arabidopsis (Col-0) dark-grown root cell suspension culture (kindly provided by Claudia Jonak, GMI, Vienna) were isolated and transfected. For transfection, we used 10 µg of reporter construct (pKB55) containing p35S:LUC (Renilla) as an internal control and 10 µg of each effector construct. The transfected protoplasts were diluted with 240 mM CaCl2 (1:3) followed by cell lysis and dual-luciferase assay using the Dual-Luciferase Reporter Assay System (Promega) and following the manufacturer’s instructions. Luminescence was measured using a Synergy H4 Hybrid Multiplate Reader (BioTek). For each reporter/effector combination, 3–5 technical replicates were done and the experiments were repeated at least three times. For experimental analysis, Firefly Luciferase activity was normalized to Renilla Luciferase activity.
Synergy h4 hybrid multiplate reader
Manufactured by Agilent Technologies
The Synergy H4 Hybrid Multiplate Reader is a versatile laboratory instrument that combines multiple detection technologies, including absorbance, fluorescence, and luminescence, in a single platform. It is designed to perform a wide range of assays, including cell-based, biochemical, and molecular biology applications.
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3 protocols using synergy h4 hybrid multiplate reader
1
Transient Reporter Activity Assays in Arabidopsis Protoplasts
2
Tubulin Polymerization Kinetics by Light Scattering
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The protocol for the light scattering experiment was adapted from44 (link). 1 µM recombinant protein was mixed with 2.5 µM tubulin and 1 mM GTP in polymerization buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA and 10% glycerol) in a total volume of 200 µl in a 96-well plate with flat bottom. The absorbance at 340 nm and 37 °C was measured for up to 2:15 hrs in a BioTek Synergy H4 Hybrid Multiplate reader. Data was collected every 38 seconds.
3
Transcriptional Repression Activity Assay
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To evaluate transcriptional repression activity, we used a standard luciferase assay. 9 23 26 Mutations were introduced into the full length coding sequence of FOXP1 longest isoform (FOXP1a; obtained from Kazusa DNA Research Institute, Kisarazu, Japan) and subcloned into the mammalian expression vector pcDNA4His (Invitrogen). HEK293 cells were then cotransfected using Fugene 6 (Roche), in 24-well plates, with 400 ng of pcDNA4His without an insert or containing the wild-type FOXP1 (wt-FOXP1) or the mutant FOXP1 cDNA, along with 50 ng of pGL3-promoter construct (Promega) (in which the SV40 promoter drives a firefly luciferase reporter). To normalise for transfection efficiency and variation in cell number, cells were also cotransfected with 50 ng of a Renilla luciferase construct (pRL-TK; Promega) driven by the HSV-thymidine kinase promoter which is not affected by FOXP1. Cells were lysed 48 hours after transfection. Firefly and Renilla luciferase activities were quantified using the Synergy H4 Hybrid Multiplate Reader (BioTek). Protein expression was verified by western blotting (see below). Student's t-test was performed to evaluate statistical significance.
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