Total proteins of transgenic soybean root nodules were extracted and quantified as previously described (Freitas et al., 2019 (link)). GUS assay was performed in a mixture containing 10 mM of 4-methylumbelliferyl-β-D -glucuronide (MUG; Sigma, United States), and the mixture was incubated for 1 h at 37°C. The fluorescence product of 4-methylumbelliferone (4-MU) was monitored using a VersaFluor Fluorometer (Bio-Rad) with excitation at 365 nm and emission at 455 nm. GUS activity was calculated in picomoles of MU produced per minute per microgram of soluble protein. The assay was repeated at least three times. The results were shown as the mean of independent experiments with the respective standard deviation. Asterisks (∗∗) above the bars indicate significant differences at p < 0.01.
4 methylumbelliferyl β d glucuronide mug
Manufactured by Merck Group
Sourced in United States
4-methylumbelliferyl-β-D-glucuronide (MUG) is a chemical compound used as a substrate in various biochemical and microbiological assays. It is a fluorogenic substrate that can be used to detect the presence of the enzyme β-glucuronidase. When β-glucuronidase cleaves the glucuronide bond in MUG, it releases a fluorescent product, 4-methylumbelliferone, which can be detected using a fluorometer.
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5 protocols using 4 methylumbelliferyl β d glucuronide mug
1
Quantification of GUS Activity in Transgenic Soybean Roots
2
Fluorometric and Histochemical GUS Assays
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GUS assays were performed as described by Jefferson et al. (1987) (link). The fluorometric assay was carried out using 4-methylumbelliferyl-β-D -glucuronide (MUG; Sigma–Aldrich, St. Louis, MO, United States) as substrate, and the histochemical staining was performed using X-Gluc (Sigma–Aldrich, St. Louis, MO, United States) as substrate.
3
Quantifying GUS Activity in Arabidopsis
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The substrate 4-methylumbelliferyl-β-d-glucuronide (MUG) (Sigma) was used to assay the GUS activity59 (link). Roots of the seedlings were frozen in liquid nitrogen and ground in a 300 μl MUG extraction buffer composed of sodium phosphate (50 mM, pH 7.0), 10 mM EDTA (pH 8.0), 10 mM β-mercaptoethanol, 0.1% (v/v) Triton X-100 and 0.1% (w/v) N-lauroyl sarcosine (SLS) (Sigma). The extract was spun, and the supernatant was extracted. 10 μl of the extract was mixed with 390 μl of the GUS assay buffer and incubated at 37°C for 1 h. The samples were stopped with 0.2 M Na2CO3. Fluorescence was determined by DyNA Quant 200. The protein concentration was measured according to Peterson's60 modification of the Lowry method. Approximately 40 seedlings were subjected to each treatment.
4
Analyzing GmPAP4 Expression in Soybean Nodules
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To elucidate the expression pattern of GmPAP4 in soybean nodules, we cloned 2000 bp promoter sequences of GmPAP4 upstream of the translation start codon ATG, fused with β-glucuronidase (GUS) reporter gene (pGmPAP4:GUS). The resulting construct was introduced into the soybean hairy roots by Agrobacterium rhizogenes K599 and the transgenic hairy roots were then inoculated with rhizobia Bradyrhizobium diazoefficiens USDA110 for nodule development. After 4 weeks of rhizobia inoculation, transgenic hairy roots and nodules were stained as described previously and captured with a light microscope (Olympus U-TV0.5XC-3, Tokyo, Japan) [14 (link)]. For GUS activity, total nodule proteins were extracted and incubated in a mixture containing 10 mM 4-methylumbelliferyl β-D-glucuronide (MUG; Sigma Chemical Co., St.Louis, MO, USA) for 1 h at 37 °C. The fluorescence product of 4-methylumbelliferone (4-MU) was monitored using a VersaFluor™fluorometer (Bio-Rad, Hercules, CA, USA) with excitation at 365 nm and emission at 455 nm.
5
Quantitative Analysis of β-Glucuronidase Activity
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Tissue or cell pellets were lysed in MUG lysis buffer (0.2M Sodium acetate buffer, pH 4.5;10 mM EDTA; 0.1% Trixton-X-100) to obtain total cell homogenate. Equal concentrations of serially diluted lysate (in the range of 0-60 μg/ml) from control and Thap1 cKO genotypes was incubated with 10 mM 4-methylumbelliferyl β-D-glucuronide (MUG, Sigma) in 0.2M Sodium acetate buffer, pH 4.5 at 37°C for one hour. The reaction was stopped with equivalent volume of 0.2M sodium citrate followed by which the fluorescence (excitation = 360nm; emission = 460nm) was measured using Biotek Synergy HT microplate reader. Based on fluorescence reading from purified βglucuronidase standard (Sigma), we determined the β-glucuronidase activity / hour and β-glucuronidase activity / mg of the lysate.
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