10 µM of HR2P-FL was incubated with 10 µM of HR1P, 5-HB or NusA-5HB in pH 7.5 20 mM PBS at 37 ◦C for 30 min. After incubation, the sample was mixed with Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA) at a ratio of 1:1 and was then loaded to 4%∼20% pre-cast gel (20 μL each well). Gel electrophoresis was carried out with 100 V constant voltages at room temperature for 1.5 h. The gel was imaged with a Tanon 1220 Gel Imaging System (Shanghai, China)。
Tris glycine native sample buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Tris-Glycine Native Sample Buffer is a buffer solution used in native polyacrylamide gel electrophoresis (native PAGE) to prepare samples for analysis. It maintains the native, non-denatured structure of proteins during electrophoresis, allowing for the separation and study of protein complexes and their interactions.
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Lab products found in correlation
Tris glycine native running buffer, by Thermo Fisher Scientific (4 mentions)
Novex tris glycine 4 20 gels, by Thermo Fisher Scientific (2 mentions)
Fluor chem 8800 imaging system, by Bio-Techne (1 mentions)
Coomassie blue, by Thermo Fisher Scientific (1 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Ni nta chromatography, by Qiagen (1 mentions)
Page gel silver staining kit, by Solarbio (1 mentions)
N dodecyl β d maltoside ddm, by Merck Group (1 mentions)
Nupage tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Esterase from porcine liver, by Merck Group (1 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Seeblue pre stained protein standard, by Thermo Fisher Scientific (1 mentions)
7 protocols using tris glycine native sample buffer
1
Protein-Protein Interaction Analysis
2
Inhibition of HIV-1 gp41 6-HB Formation
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The inhibition of gp41 6-HB formation by NSPD-12m was further confirmed by N-PAGE as reported previously (Liu et al., 2005 (link)). Briefly, NSPD-12m at the indicated concentrations was incubated with the peptide N36 (100 μM in PBS) at 37°C for 30 min before the addition of the peptide C34 (100 μM in PBS). After incubation at 37°C for another 30 min, the mixture was diluted in Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA) and then loaded onto 10 × 1.0-cm precast Tris-glycine gels (18%, Invitrogen) at 20 µl/well. Gel electrophoresis was carried out at constant voltage of 120 V at room temperature (RT) for 2 h in Tris-glycine native running buffer. Immediately after electrophoresis, the gel was stained by Coomassie blue (Invitrogen) and imaged by the FluorChem 8800 Imaging System (Alpha Innotech, San Leandro, CA). ADS-J1 (1 mM in PBS), a small-molecule HIV-1 entry inhibitor that blocks gp41 6-HB formation, was used as a positive control (Wang et al., 2009 (link)).
3
Production and Purification of SARS-CoV-2 Spike Protein
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To express the prefusion Spike (S) ectodomain, a gene encoding the residues 1–1208 of 2019-nCoV S (GenBank: MN908947) with proline substitutions at residues 986 and 987, a “GSAS (amino acid sequence)” substitution at the furin cleavage site (residues 682–685), a C-terminal T4 fibritin trimerization motif, and an 8XHisTag were synthesized and cloned into the pcDNA3.1 vector. The plasmid was transfected to 293T cells and the recombinant S protein trimers were purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography (QIAGEN, Hilden, Germany), followed by size exclusion to further purify the trimers. The trimeric Spike protein was dissolved in a Tris-glycine native sample buffer (Thermo Fisher, Waltham, MA, USA), and separated by a 3%–8% Tris-acetate protein gel under a non-reducing condition. The trimeric Spike protein was visualized by a PAGE gel silver staining kit (Solarbio, Beijing, China). The monomeric Spike protein was produced as a secreted form by BTI-Tn-5B1-4 insect cells as described by the authors of [55 (link)].
4
BN-PAGE Gel Shift Assay for Env Variants
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BN-PAGE gel mobility shift assays were performed using virions produced by transfection of the infectious molecular clone plasmid pLAI displaying the Env variant ADA.CM (Leaman and Zwick, 2013 (link)). Virions and a dilution series of Fab were incubated at RT for 30 min. The virion/Ab mixture was solubilized in 1% n-Dodecyl β-D-maltoside (DDM; Sigma) for 20 min on ice, and then run on a 3–8% gradient Tris-Acetate NuPAGE gel (ThermoFisher) in Tris-Glycine Native Sample Buffer (ThermoFisher), supplemented with 0.25% Coomassie G-250. Gels were run for 3 h at 150 V in Tris-Glycine Native Running Buffer (ThermoFisher) + 0.002% Coomassie G-250. Proteins were transferred onto PDVF membrane and Western blotted using a cocktail of primary antibodies to gp120 (F105, 2G12 and HGN194, 2 μg/mL each) and gp41 (10E8, 2F5, and 7B2, 1 μg/mL each) followed by a goat anti-human-Fcγ-HRP secondary antibody (Jackson). The blot was developed using ECL Plus Substrate (Pierce) and a ChemiDoc XRS + Imaging System (Bio-Rad).
5
Esterase Activity Assay in Bacterial Spores
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Proteins washed from 220 mg of spores were freeze-dried, then dissolved in 500 μL of 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 1 mM EDTA, 1% v/v Triton X-100, 0.1 M PMSF, and a protease inhibitor cocktail tablet (Roche Diagnostics). Intracellular proteins were isolated from 220 mg of washed spores by grinding in liquid nitrogen. The tissue powder was dissolved in 500 μL of the same buffer as the extracellular proteins. The lysate was clarified by centrifugation (16,900 × g, 4°C, 20 min), and the supernatant was collected. Protein concentration was determined using the Bradford protein assay. Esterase from porcine liver (Sigma) was used as a positive control of hydrolysis.
Proteins were separated using a Novex Tris-Glycine gel (Thermo Fisher Scientific) and Tris-Glycine Native Sample Buffer (Thermo Fisher Scientific). Preparations containing 1.0, 0.1, or 0.05 μg proteins were loaded onto the gel. SeeBlue Pre-stained Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker. Electrophoresis was conducted at constant 125 V for 3 h at 4°C in 1× Tris-glycine native running buffer (Thermo Fisher Scientific). The gel was then washed twice for 10 min each in 100 mM Tris-HCI, pH 8.0. Esterase activity was detected using the indoxyl acetate assay as described [50 (link)].
Proteins were separated using a Novex Tris-Glycine gel (Thermo Fisher Scientific) and Tris-Glycine Native Sample Buffer (Thermo Fisher Scientific). Preparations containing 1.0, 0.1, or 0.05 μg proteins were loaded onto the gel. SeeBlue Pre-stained Protein Standard (Thermo Fisher Scientific) was used as the molecular weight marker. Electrophoresis was conducted at constant 125 V for 3 h at 4°C in 1× Tris-glycine native running buffer (Thermo Fisher Scientific). The gel was then washed twice for 10 min each in 100 mM Tris-HCI, pH 8.0. Esterase activity was detected using the indoxyl acetate assay as described [50 (link)].
6
Heme Transfer Assay for PGRMC2
7
Heme Transfer Assay for PGRMC2
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Heme transfer was assessed by mixing 10 μg of WT or mouse PGRMC2 heme-binding mutant (3xM) with 10 μg of apo-REV-ERBα protein and incubating for 30 min at 37°C. After incubation, 2X Native Tris-Glycine sample buffer (Life Technologies) was added and samples separated by electrophoresis using Novex Tris-Glycine 4-20% gels and Tris-Glycine Native Running Buffer (Life Technologies) for 6 hr. The gel was washed for 10 min with water and heme staining was performed using the BioFX TMB One Component HRP Microwell Substrate (Surmodics). After imaging the heme stain, the gel was washed overnight with water and counterstained with Coomassie for protein detection.
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