The experiment was conducted by following Lv et al., 2014 . Briefly, total protein of 10 days‐after‐germination rice seedlings were extracted in degradation buffer (25 mm Tris‐HCl, pH 7.5, 10 mm NaCl, 10 mm MgCl2, 4 mm PMSF, 5 mm DTT, and 10 mm ATP) and quantified using a Quabit system (Invitrogen, Carlsbad, CA). Purified recombinant proteins (5 μg of each) were incubated with 200 μg extracted total proteins in 20 μL degradation buffer at 28 °C. Reactions were terminated at indicated time points, and the protein abundance was visualized by immune detection against anti‐HIS. The immune signals were quantified using Quantity Tools of Image Lab software (Bio‐Rad, Hercules, CA). The half‐life of HIS‐NF‐YB1 was calculated based on the degradation curves deduced from the tested time points. The protein intensities were quantified using ImageJ software with triplicates. The dissociation‐one phase exponential decay curve was plotted on a semilog graph using Graphpad Prism (5.0) software as previously described by (Lv et al., 2014 ).
Quantity tools of image lab software
Manufactured by Bio-Rad
The Quantity Tools of Image Lab software provide a set of tools for quantifying and analyzing images. These tools allow users to measure the intensity, size, and other properties of specific regions or bands within an image. The Quantity Tools are designed to be straightforward and easy to use, enabling researchers to quickly extract meaningful data from their experiments.
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Lab products found in correlation
Quabit system, by Thermo Fisher Scientific (1 mentions)
Ubiquitinylation kit, by Enzo Life Sciences (1 mentions)
Anti gst antibody, by Transgene (1 mentions)
Goat anti mouse igg hrp, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Merck Group (1 mentions)
Immobilon kit, by Merck Group (1 mentions)
Anti his hrp, by Merck Group (1 mentions)
Anti flag m2 hrp, by Merck Group (1 mentions)
3 protocols using quantity tools of image lab software
1
Protein Degradation Kinetics in Rice Seedlings
2
Cell-free Protein Degradation Assays
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The cell-free degradation assays were performed as previously described (Lv et al., 2014) . In brief, the shoots of seedlings (WT, sdel1, sdel2, sdel1sdel2, phr1phr2 , 5 mM dithiothreitol, and 10 mM ATP) and adjusted to equal concentrations in degradation buffer for each assay. The purified proteins of GST, GST-SPX4, GST-SPX4-N, GST-SPX4-C, GST-SPX4-C K213R , GST-SPX4-C K213R/K299R , and GST-SPX4 K213R/K299R were incubated with various protein extracts at 28 C for 0, 5, 10, 20, and 40 min. The GSTtagged proteins were detected by an anti-GST antibody (TransGen Biotech) diluted 1:5000. Quantitative analysis of immunoblots was performed using the Quantity Tools of Image Lab software (Bio-Rad).
Recombinant Protein Purification and In Vitro Ubiquitination Assays SDEL1-His, PHR2-His, GST-SPX4, and SPX4-related truncates or mutated proteins were overexpressed and purified from E. coli BL21 (DE3) cell cultures using standard protocols. In vitro ubiquitination assays were performed using a Ubiquitinylation Kit (Enzo Life Sciences, BML-UW9920-0001) according to the manufacturer's instructions. The UB-CH5a E2 enzyme in the kit was used in this study. Ubiquitin, conjugated to biotin, was detected by immunoblotting with stabilized streptavidinhorseradish peroxidase (HRP) conjugate.
Recombinant Protein Purification and In Vitro Ubiquitination Assays SDEL1-His, PHR2-His, GST-SPX4, and SPX4-related truncates or mutated proteins were overexpressed and purified from E. coli BL21 (DE3) cell cultures using standard protocols. In vitro ubiquitination assays were performed using a Ubiquitinylation Kit (Enzo Life Sciences, BML-UW9920-0001) according to the manufacturer's instructions. The UB-CH5a E2 enzyme in the kit was used in this study. Ubiquitin, conjugated to biotin, was detected by immunoblotting with stabilized streptavidinhorseradish peroxidase (HRP) conjugate.
3
Western Blot Antibody Validation
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Antibodies used for western blotting were anti-GFP-HRP (G1544, Sigma, 1:5000), anti-FLAG-HRP (M2, Sigma, 1:5000), anti-His-HRP (Sigma, 1:5000), anti-GST-HRP (TransGen Biotech, 1:5000), mouse monoclonal anti-Ubi-HRP (Santa Cruz Biotechnology, 1:2000), goat anti-rabbit-IgG-HRP (Sigma, 1:10 000), and goat anti-mouse-IgG-HRP (Sigma, 1:10 000). Total plant proteins were extracted using extraction buffer (containing 25 mM Tris-HCl [pH 7.5], 10 mM NaCl, 4 mM PMSF, 20 mM MG132, and protease inhibitor cocktail). Proteins were visualized using the Immobilon kit (Millipore) under standard conditions. Quantitative analysis of immunoblots was performed using the Quantity Tools of Image Lab software (Bio-Rad).
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