Total soluble protein (TSP) was extracted as described above for fluorometric assays. A protein sample (25 µg) from each studied line was separated by 12% SDS-PAGE and transferred onto an NC membrane (BioRad, Hercules, USA). Rabbit anti-β-glucuronidase (diluted 1:2000, Sigma-Aldrich, St. Louis, USA) polyclonal antibodies served as the primary antibodies. Anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody (1:4000, Pierce, Waltham, USA). Blots were treated with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for visualization.
Rabbit anti β glucuronidase
Manufactured by Merck Group
Sourced in United States
Rabbit anti-β-glucuronidase is a laboratory reagent used for the detection and quantification of the enzyme β-glucuronidase in various biological samples. It is an antibody raised in rabbits that specifically binds to and recognizes the β-glucuronidase protein.
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Lab products found in correlation
4 protocols using rabbit anti β glucuronidase
1
SDS-PAGE Immunoblot for β-Glucuronidase
2
Protein Isolation and Western Blot
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Protein was isolated using custom made RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1mM EDTA, 1% Triton x-100) supplemented with protease inhibitor (Pierce, 78442). Concentration of isolated protein was measured using a microBCA kit (Pierce, 23227) and 25μg was loaded on a 4-12% Bis-Tris protein gel (Invitrogen, NP0321BOX). Protein size was analyzed using PageRuler™ Prestained Protein Ladder (Thermo 26616). Membranes were blocked in 5% non-fat milk in TBS-T. Antibodies used included rabbit anti-ΔNp63 antibody (Cell Signaling E6Q3O), mouse anti-p53 (BD Biosciences 554293), mouse anti-TAp63 (BioLegend 938102) rabbit anti-GAPDH antibody (Cell Signaling D16H11), and rabbit anti-β -Glucuronidase (Sigma Aldrich G5545).
3
Extraction and Analysis of Total Soluble Proteins
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To prepare total soluble protein, duckweed plants (0.5 g) were ground in liquid nitrogen. The ground material was resuspended in four volumes of extraction buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0, 10 % (v/ v) glycerol, 1 % (w/v) SDS, 30 mM 2-mercaptoethanol, 4 lg/ml aprotinin, and 4 lg/ml leupeptin. Total proteins were extracted for 20 min at 4 °C, then centrifuged for 10 min at 16,000 g, 4 °C and the supernatant was taken for further analysis. Protein concentration was measured by DC protein assay (BioRad, USA).
Total proteins (25 lg) from each transgenic line were separated by 12 % SDS-PAGE and transferred onto an NC membrane (Amersham). Rabbit anti-b-glucuronidase (diluted 1:2000, Sigma, USA) and anti-M2e (1:1000, Abcam, UK) polyclonal antibodies served as the primary antibodies. Anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody (1:4000, Pierce, USA). Blots were treated with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for visualization.
Total proteins (25 lg) from each transgenic line were separated by 12 % SDS-PAGE and transferred onto an NC membrane (Amersham). Rabbit anti-b-glucuronidase (diluted 1:2000, Sigma, USA) and anti-M2e (1:1000, Abcam, UK) polyclonal antibodies served as the primary antibodies. Anti-rabbit IgG conjugated to alkaline phosphatase was used as the secondary antibody (1:4000, Pierce, USA). Blots were treated with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) for visualization.
4
Quantification of Recombinant Enzyme Expression
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Total soluble protein (TSP) was extracted as described above in extraction buffer without SDS. The protein samples were serially diluted in phosphate buffer saline (PBS) (0.25, 0.5, 1.0, and 2.0 lg TSP per well) and placed into a 96-well microtiter plate, using b-glucuronidase from E. coli (Sigma) as the reference standard. The plates were incubated for 2 h at room temperature, washed three times with washing buffer for 5 min each [PBS plus 0.05 % (v/v) Tween-20] and blocked with blocking buffer (PBS containing 2 % w/v bovine serum albumin and 0.05 % Tween-20) for 1 h at 37 °C. The plates were then incubated with rabbit anti-bglucuronidase (diluted 1:1000, Sigma) polyclonal antibody overnight at 4 °C. After washing, the plates were incubated with anti-rabbit IgG conjugated to alkaline phosphatase (1:3000, Pierce) for 1 h at 37 °C. The plates were washed and developed by the addition of 100 ll per well of TMB Peroxidase EIA substrate (BioRad) for 30 min at room temperature. The plate was read at 405 nm and the amount of plant-expressed M130-b-glucuronidase was estimated based on reference b-glucuronidase standards.
To determine the expression level of M130-b-glucuronidase in the transgenic lines, three samples of duckweed per line were analyzed, and their average expression level calculated. All measurements were performed in duplicate.
To determine the expression level of M130-b-glucuronidase in the transgenic lines, three samples of duckweed per line were analyzed, and their average expression level calculated. All measurements were performed in duplicate.
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