The vectors containing GUS driven by different promoters were transformed into rice protoplasts with or without SIP1. After incubation at 25°C for 12 h, the protoplasts were lysed with lysis buffer (25 mM Tris‐HCl, pH 7.8, 1 mM DTT, 10% glycerol and 1% Triton X‐100). After centrifugation, 100 μl of supernatant was mixed with 900 μl of fluorescent β‐galactosidase (MUG) substrate mix (10 mM Tris‐HCl, pH 8.0, containing 1 mM MUG and 2 mM MgCl2). After incubation at 37°C for 30 min, the reaction was stopped with 40 mM Na2CO3. GUS activity was measured using a fluorometer (HITACHI, U‐281; Thermo Fisher, Fluoroskan Ascent FL, Waltham, MA, USA) with 365 nm excitation wavelength and 456 nm emission wavelength. The luciferase (LUC) activity was measured using the GloMax 96 Luminometer system (Promega, E6501) with LUC mix (Promega, E1980).
Glomax 96 luminometer system
Manufactured by Promega
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The GloMax 96 Luminometer system is a compact and sensitive instrument designed for the measurement of luminescent signals in microplate format. It provides accurate and reliable quantification of various luminescent assays, including reporter gene assays, cell-based assays, and biochemical assays.
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2 protocols using glomax 96 luminometer system
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GUS Activity Assay in Rice Protoplasts
2
GUS and LUC Activity Assay in Arabidopsis Protoplasts
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The constructs containing GUS driven by the OST1, SnRK2.2, and SnRK2.3 promoters were transformed into Arabidopsis protoplasts and incubation at 25°C for 12 h. The protoplasts were then transferred into lysine buffer (25 mM Tris-HCl pH 7.8, 1 mM DTT, 10% glycerol, and 1% Triton X-100). Supernatant (100 µL) was mixed with 900 µL of MUG substrates (10 mM Tris-HCl pH 8.0, containing 1 mM MUG and 2 mM MgCl 2 ). This reaction was performed at 37°C for 30 min and stopped with 40 mM Na 2 CO 3 . GUS activity was measured using a fluorometer (HITACHI, Tokyo, Japan; U-281) with 365 nm excitation wavelength and 456 nm emission wavelength. The LUC activity was measured using the GloMax 96 Luminometer system (Promega, Madison, Wisconsin, USA; E6501) with LUC mix (Promega, E1980).
For the promoter GFP assay, leaves from 7-day-old seedlings were scanned using confocal microscopy (Zeiss, Oberkochen, Germany, LSM 980).
For the promoter GFP assay, leaves from 7-day-old seedlings were scanned using confocal microscopy (Zeiss, Oberkochen, Germany, LSM 980).
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