Cells were lysed with reporter lysis buffer (1×, Promega) supplemented with protease inhibitor (cOmplete, Mini, EDTA-free tablets; Roche) and phosphatase inhibitor (PhosStop Phosphatase Inhibitor Cocktail; Roche). Proteins were separated on 10% SDS-PAGE gels, transferred to PVDF membrane, and probed with primary antibodies at room temperature for 2 hours or at 4°C overnight. After incubation with secondary antibodies and development, bands were detected on film (Biomax MR film; Carestream Health) or using the ChemiDoc Imaging System (Bio-Rad) and quantified using Image Studio Lite software (Li-COR) or Image Lab software (Bio-Rad).
Complete mini edta free tablet
Manufactured by Roche
Sourced in Switzerland, United States
Complete Mini EDTA-free tablets are a laboratory reagent used for protein extraction and purification. They are designed to disrupt cell membranes and release proteins while avoiding the chelating effects of EDTA.
Automatically generated - may contain errors
Lab products found in correlation
Duoset kit, by R&D Systems (2 mentions)
Near infrared fluorescent secondary antibodies, by LI COR (2 mentions)
0.45 μm nitrocellulose membrane, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Phosstop, by Roche (2 mentions)
Lowry assay, by Bio-Rad (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Reporter lysis buffer 1, by Promega (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Phosstop phosphatase inhibitor cocktail, by Roche (1 mentions)
Biomax mr film, by Carestream (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Nebuffer 2, by New England Biolabs (1 mentions)
29 protocols using complete mini edta free tablet
1
Western Blot Protein Quantification
2
Chromatin Digestion and Hi-C Library Preparation
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Each cross-linked cell aliquot (∼20 million cells) was resuspended in 50 mL of permeabilization buffer (10 mM Tris-HCl at pH 8, 10 mM NaCl, 0.2% IGEPAL CA-630 [Sigma-Aldrich], supplemented with complete mini EDTA free tablets [Roche]) and incubated on ice for 30 min with occasional mixing. SUM44 and GM06990 cells were lysed using 10 strokes of a dounce homogenizer. BT483 and MCF7 cells were lysed by incubating with trypsin (0.25%, Sigma-Aldrich) at 37°C for 5 min. trypsin was inactivated by addition of 500 µL FBS. Permeabilized cells were centrifuged for 6 min at 600g and washed three times in 1 mL 1.3 × NEBuffer 2 (New England Biolabs). Nuclei were resuspended and chromatin digestion and Hi-C library preparation were carried out as described by van Berkum et al. (2010) with the following modifications: (1) Cells were split into three microcentrifuge tubes instead of five; (2) restriction fragment overhangs were filled in with biotinylated dATP instead of biotinylated dCTP; (3) dGTP was added to the reaction mixture for the removal of biotinylated dATP from unligated ends; (4) we did not include an agarose gel size selection step; (5) after PCR amplification (six to eight cycles) of the Hi-C library bound streptavidin beads, the PCR product was pooled and subjected to target enrichment (below) before paired-end sequencing.
3
HA-Immunoprecipitation from HEK-293T Cells
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Following infection and expansion, HEK-293T cells were trypsinized, centrifuged, and resuspended with lysis buffer, which was composed of 50 mM Tris (pH 7), 100 mM NaCl, 1 mM EGTA, 1% Triton-X 100, and a protease inhibitor cocktail at 1 tablet/50 ml (complete mini-EDTA-free tablets from Roche, Indianapolis, IN). The lysate was incubated for 20 min on ice and then centrifuged at 20,000×g for 10 min. The supernatant was used for immunoprecipitations (IPs). Protein G Dynabeads (Novex, Carlsbad, CA) were used with a magnetic bead separator (Invitrogen, Carlsbad, CA). For the IP, 50 µl of Dynabeads and 5 µg of primary antibody were incubated for 10 min at room temperature. The primary antibodies included rabbit anti-HA antibody (Rockland, Gilbertsville, PA) or mouse anti-HA antibody (Millipore, Billerica, MA). The beads were then washed with PBST once, followed by incubation with cell lysate at room temperature for 30 min. The Dynabeads were washed three times with 50 mM Tris (pH 7), 100 mM NaCl, and 0.1% Tween-20 and finally resuspended with 24 µl of 1× loading buffer. Equal volumes of samples were analyzed by SDS-PAGE.
4
Bronchoalveolar Lavage Fluid Cytokine Assay
5
Lung Lavage and Cell Analysis
6
Protein Extraction and Immunoblotting
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Cells were washed twice with 1× PBS and lysed on ice for 30 min in 1× lysis buffer, containing 150-mm NaCl, 10-mm Tris–HCl pH 6.8, 1-mm EDTA pH 8.0, 10% glycerol and 1% Triton X-100, and supplemented with a protease inhibitor cocktail (cOmplete-mini EDTA-free tablets-Roche) and, when appropriate, with phosphatase inhibitors (Sigma-Aldrich). After centrifugation for 15 min at 14 000 rpm at 4°C, cleared lysates were obtained. Sample proteins were separated using 12% SDS-PAGE gels, followed by their transfer onto PVDF and detection using primary antibodies (1:1000) followed by incubation with HRP mouse or rabbit IgG (1:5000) (GE Healthcare Amersham, Little Chalfont, UK) in PBS containing 5% non-fat dry milk (Bio-Rad Laboratories). In all the experiments, unless otherwise indicated, the volumes of both lysis buffer and conditioned media used were kept constant (namely, 600 μL for the six multi-well format and 1500 μL for the 10-cm dish format) and the same ratio between intracellular and extracellular pool samples was used (1:10 IN/OUT ratio, volume/volume) in order to allow and optimize the detection of even small amounts of secreted proteins.
7
Anther Protein Extraction and Western Blot
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Anther proteins were extracted in 150 mm NaCl, 10% glycerol, 2 mm EDTA and Tris-HCl, pH 7.5, containing protease inhibitors (Complete mini EDTA-free tablets; Roche, http://www.roche.com ) and insoluble material was removed by centrifugation. Protein samples were separated by SDS-PAGE and western blotted as described by Armstrong et al. (2002 (link)). Anti-AtSMC4 antibody was used at a dilution of 1/1000.
8
Lysis and Protein Extraction from PDX Samples
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Cell lysis of snap-frozen patient-derived xenografts (PDX) was performed with a lysis buffer (6 M urea, 2 M thiourea, 10 mM Tris-HCl pH 8.0) supplemented with a protease inhibitor (complete Mini EDTA-free tablets; Roche, Basel, Switzerland) and phosphatase inhibitor buffers (5 mM glycerol-2-phosphate, 5 mM sodium fluoride, and 1 mM sodium orthovanadate). Glass beads (zirconia/glass beads 0.23 mm; Carl Roth GmbH, Karlruhe, Germany) were added and a cell lysis was performed in a BeadBug microtube homogenizer (3 cycles, 1 min at full speed; Sigma-Aldrich, St. Louis, MO, USA). Cell extracts were centrifuged at 13,000 rpm for 20 min and proteins were purified by acetone precipitation. Briefly, cell lysates were mixed with 8 volumes of ice-cold acetone and one volume of methanol and incubated overnight at −21°C. After centrifugation (2800× g, 20 min, 10 °C), protein pellets were washed with 80% acetone and dissolved in a lysis buffer. The protein concentration was determined by a Bradford assay.
9
Protein Extraction from Biological Samples
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Samples were thawed on ice and lysed in Complete Mesoscale Lysis Buffer (pH 7.5; 150 mM sodium chloride (Sigma-Aldrich, cat. no. 1064041000), 20 mM Tris, 1 mM Ethylene Diamine Tetra Acetate (EDTA, Sigma-Aldrich, cat. no. E9884), 1 mM ethylene glycol tetraacetic acid (EGTA, Sigma-Aldrich cat. no. E43781% Triton-X-100 (Merck, cat. no. X100), a cocktail of phosphatase and proteinase inhibitors (Sigma-Aldrich, cat. no. P5726 and Sigma-Aldrich, cat. no. P0044), and Complete, Mini, EDTA-free tablets (Roche, cat. no. 11836170001)), and tip-sonicated. After being shaken on ice at 4 °C for 30 min, samples were centrifuged at 14,000× g at 4 °C for 20 min, and the supernatants were stored at −80 °C until further analysis. The protein concentration was determined using the Pierce Bicinchoninic acid (BCA) Protein Assay Kit (ThermoFischer Scientific, cat. no. 23235) according to the manufacturer’s protocol.
10
Molecular Signaling in Alzheimer's Disease
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Tissues from 3xTg‐AD mice and AD patients were homogenized with a douncer and sonicated in 200 μl of RIPA with a protease inhibitor cocktail (Complete, Mini EDTA‐free tablets, Roche, Mannheim, Germany). Astrocyte (1×105) and tissue homogenates were centrifuged at 4°C for 10 min at 12,000 × g, and protein content in the supernatant was quantified with the Bio‐Rad Protein Assay (Bio‐Rad, Hercules, CA, USA). Cell and tissue extracts (10 μg) were loaded into gels and blots were developed with rabbit polyclonal anti‐integrin β1, antiphosphoPAK, anti‐PAK, antiphosphoPKC, antiphosphoPKD, antiphosphoAKT, anti‐AKT (1:1000, Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti‐NOX2, anti‐NOX‐1 (1:1000, Novus Biologicals, Littleton, CO, USA), rabbit polyclonal anti‐β actin (1:5000, Sigma, St. Louis, MO, USA), mouse monoclonal anti‐GFAP, anti‐Rac1, anti‐GAPDH (1:2000, Millipore Ibérica, Madrid, Spain), and anti‐beta amyloid 1–16 (1:1000, 6E10 BioLegend, San Diego, CA, USA). For dot blot assay, human tissue extracts (2 μg) were spotted into a nitrocellulose membrane, and rabbit polyclonal anti‐Aβ1‐42 (1:10000, Abcam, Cambridge, UK) was used for chemiluminescence detection.
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