A luciferase (LUC) reporter activity driven by deletion and mutated CYP707A2 promoters were tested in a protoplast transient assay. 35S::NLP8-GFP and 35S::GUS (pCAMBIA1105.1R) were used as an effector and an internal control plasmid, respectively. Transfection-grade plasmids were isolated by FastGene Xpress Plasmid PLUS Kit (FastGene). Arabidopsis protoplasts were prepared from young rosette leaves of 3-week-old plants as previously described42 (link). A reporter (7.5 μg), an effector (7.5 μg) and an internal control GUS plasmid (1 μg) were co-transfected into ∼2 × 104 protoplasts by a 20% w/v polyethylene glycol 4000-mediated method. In the non-effector experiment, the pMD20 (7.5 μg) plasmid containing ACT2 (AT3G18780) cDNA was used to adjust total amount of input DNA as a control. After transfection, cells were cultured with 0.5 M mannitol and 4 mM MES (pH 5.7) containing 20 mM KCl or KNO3 for 18 h at 22 °C. Cells were collected, and then LUC and GUS activities were measured using luciferase assay system kit (Promega) and 4-methylumbelliferyl-D -glucuronide (Sigma), respectively, as substrates. The relative promoter activity was determined by calculating ratios of LUC and GUS activities.
4 methylumbelliferyl d glucuronide
Manufactured by Merck Group
Sourced in United States
4-methylumbelliferyl-D-glucuronide is a chemical compound used as a substrate in various analytical and diagnostic applications. It is a fluorogenic substrate that can be used to detect the presence of the enzyme beta-glucuronidase. The compound exhibits fluorescence upon enzymatic hydrolysis, allowing for the quantification of beta-glucuronidase activity.
Automatically generated - may contain errors
3 protocols using 4 methylumbelliferyl d glucuronide
1
Protoplast Transient Assay to Analyze CYP707A2 Promoter
2
Serum GUSB Activity Measurement
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Blood serum was collected by retroorbital sinus puncture from chimeric mice and age-matched controls. Serum GUSB activity was measured using 4-methylumbelliferyl-d-glucuronide (Sigma, USA) as substrate, described previously (Shapira et al. 1989 ) with some modification.
3
Quantifying Tomato Transcription Factor Activity
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Proteins were separated by SDS-PAGE, were transferred to nitrocellulose, and were then detected by ECL prime (GE) using anti-GFP (Covance) and horseradish peroxidaseconjugated secondary antibodies (Bio-Rad). Primary and secondary antibodies were typically diluted 1:5,000 and 1:3,000, respectively.
Tomato GUS reporter assays.
To monitor effector induction of bHLH132 transcription, A. tumefaciens 1D1249 cells harboring effector plasmids [pEAQ-HT-DEST2(6xHis-effector)] were inoculated into leaves as described above. After 72 h, leaf discs (0.6 cm 2 ) were ground in 200 µl of GUS extraction buffer (50 mM sodium phosphate, pH 7, 10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100, and 10 mM b-mercaptoethanol) and were centrifuged at 16,000 × g for 10 min at 4°C. Supernatant (10 µl) was mixed with 90 µl of GUS extraction buffer with 1 mM 4-methylumbelliferyl-D-glucuronide (Sigma-Aldrich). The mixture was incubated at 37°C for 1 h. GUS activity was determined using the Wallac 1420 multilabel counter (Perkin Elmer). Total protein concentration was determined using Bradford protein assay.
Tomato GUS reporter assays.
To monitor effector induction of bHLH132 transcription, A. tumefaciens 1D1249 cells harboring effector plasmids [pEAQ-HT-DEST2(6xHis-effector)] were inoculated into leaves as described above. After 72 h, leaf discs (0.6 cm 2 ) were ground in 200 µl of GUS extraction buffer (50 mM sodium phosphate, pH 7, 10 mM EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100, and 10 mM b-mercaptoethanol) and were centrifuged at 16,000 × g for 10 min at 4°C. Supernatant (10 µl) was mixed with 90 µl of GUS extraction buffer with 1 mM 4-methylumbelliferyl-D-glucuronide (Sigma-Aldrich). The mixture was incubated at 37°C for 1 h. GUS activity was determined using the Wallac 1420 multilabel counter (Perkin Elmer). Total protein concentration was determined using Bradford protein assay.
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