Cell pellets were resuspended in lysis buffer containing 100 mM Hepes pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 2 mM PMSF, and 1× complete EDTA-free protease inhibitor cocktail (Roche, Millipore Sigma). Lysates were mixed thoroughly by pipetting up and down with 1-mL Eppendorf pipette tips and then allowed to sit on ice for 30 min. Cellular debris was removed by centrifugation at 14,000g for 30 min at 4 °C. Protein concentrations were determined by bicinchoninic acid (BCA) assay (Bio-Rad), and cell lysates were diluted with lysis buffer to normalize final protein concentrations, typically to ∼3 mg/ml. Portions of the samples to be reacted with biotin-azide for affinity purification using the original OPP-ID protocol (16 (link)) were precleared by overnight incubation with streptavidin agarose resin (Thermo Scientific) at 4 °C with slow rotation prior to click chemistry.
Bicinchoninic acid bca assay
Manufactured by Bio-Rad
The Bicinchoninic acid (BCA) assay is a colorimetric detection and quantification method used to measure the total protein concentration in a sample. The assay is based on the reduction of copper ions (Cu2+ to Cu+) by proteins in an alkaline medium, and the subsequent chelation of the cuprous cation (Cu+) by two molecules of BCA, resulting in a purple-colored complex that absorbs light at 562 nm. This method is widely used in various applications, including biochemical research, protein quantification, and clinical diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin agarose resin, by Thermo Fisher Scientific (1 mentions)
Pipette tip, by Eppendorf (1 mentions)
Complete edta free protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Ni nta, by Qiagen (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Cell disrupter, by Constant Systems (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
7 protocols using bicinchoninic acid bca assay
1
Cell Lysis and Protein Normalization
2
Western Blot Analysis of Epigenetic Regulators
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The cells were lysed with 500 mL of Radioimmunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors (Millipore). Bio‐Rad's Bicinchoninic Acid (BCA) assay was used to measure protein concentrations. Proteins were separated using 6% and 10% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gels and then transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) in Tris Buffered Saline (TBS) for 1 h at room temperature. They were then incubated overnight at 4°C with the primary antibody. Afterward, the membranes were washed with TBS containing 1% Tween 20 and incubated with an Horseradish Peroxidase (HRP)‐conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were developed using an Enhanced chemiluminescence (ECL) reagent (Thermo). The immunoblots were quantified by Bio‐Rad Quantity One version 4.1 software. Primary antibodies against SETD2 (LS‐C332416), histone H3 (trimethylK36) (ab9050), Smad7 (ab216428) and FN (15613) were purchased from Lifespan, Abcam and Proteintech. Antibodies against histone H3 (#9715), p‐Smad3 (#9520), Smad3 (#9523), p‐Smad2 (#3108), Smad2 (#5339) and COL1A1 (#72026) were purchased from Cell Signaling Technology Inc.
3
Protein Extraction from Vascular Tissues
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Ascending aorta and most branches of mesenteric arteries were dissected after flushing the vasculature with 1× PBS through the apex of the left ventricle. Tissues were stored at −80°C until protein extraction was performed. Tissues were homogenized using TissueLyser II (Qiagen) at 30 Hz for 5 min in an 8 M urea solution in 16 mM Na2HPO4 (pH 7) containing protease inhibitors, and homogenates were incubated at 4°C overnight. After centrifugation, the supernatant was diluted to 2 M urea using 16 mM Na2HPO4, and bicinchoninic acid (BCA) assay (Bio-Rad) was performed to measure protein concentration. Protein was then precipitated using 10% trichloroacetic acid at 4°C for 1 hour. After centrifugation, the pellet was washed three times with ice-cold acetone and resuspended in Laemmli buffer containing dithiothreitol (DTT). The suspension was boiled for 5 min and used for SDS–polyacrylamide gel electrophoresis (SDS-PAGE).
4
Purification and Characterization of SaeR-His6 Protein
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The SaeR-His6 protein was overproduced in E. coli BL21(DE3) harboring pET22b-saeR. Overnight cultures were inoculated into fresh LB broth, and SaeR-His6 was overexpressed by the addition of 1 mM isopropyl-1-thio-β-d -galactopyranoside (IPTG) to the fresh culture. The bacterial culture was further incubated at 37°C for 6 h. SaeR-His6 was purified with nickel-nitrilotriacetic acid (Ni-NTA) column chromatography (Qiagen) by following the manufacturer’s recommendations. The purified SaeR-His6 protein was exchanged with TBS buffer (10 mM Tris-HCl [pH 7.5], 138 mM NaCl, 2.7 mM KCl), followed by TBS buffer containing 25% (vol/vol) glycerol, and concentrated using an Amicon Ultra-15 centrifugal filter unit (molecular weight [MW] cutoff of 10,000; Millipore). The MBP-SaeS protein was purified as described before (20 (link)). Protein concentration was determined by the bicinchoninic acid (BCA) assay (Bio-Rad), and the purified proteins were stored at −80°C until used.
5
Isolation and Analysis of Membrane Vesicles
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Membrane vesicles were isolated as previously described.18 (link),21 (link) Briefly, cells carrying pBlueScript-based QacA variants were lysed by one passage through a cell disrupter (Constant Systems) at 30 000 psi. Membrane vesicles were isolated by centrifugation (134 000 × g, 1 h, 4°C) and resuspended in buffer [20 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10% (v/v) glycerol]. Protein concentrations were quantified using a bicinchoninic acid (BCA) assay (Bio-Rad). Equal amounts of protein were loaded onto a 10% SDS-PAGE gel and western blot analysis was performed using a primary rabbit anti-6 × His-tag antibody (Rockland Immunochemicals, 1:5000 dilution) and a secondary goat-anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad, 1:10 000 dilution).17 (link),20 (link) Blots were scanned using a ChemiDoc™ MP Imaging System (Bio-Rad) and analysed by Image Lab software (Bio-Rad).
6
Intracellular GTP Quantification Using GTPase-Glo
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A GTPase-Glo bioluminescence assay (Promega) was used, and manufacturer’s guidelines were adjusted to measure relative intracellular GTP levels by luminescence. Bacterial cultures were pelleted and resuspended in TSM buffer (50 mM Tris pH 7.5, 0.5 M sucrose, 10 mM MgCl2) before lysing with 50 μg/mL lysostaphin and 50 μg/mL DNase for 30 min at 37°C. Total protein concentrations were determined using a Bicinchoninic Acid (BCA) assay (Bio-Rad). The protein concentration of each lysate was adjusted to 100 μg/mL and 2.5 μL (250 ng) used in each GTPase-Glo assay.
7
Protein Expression Analysis in Thyroid Cell Lines
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PTC and ATC cell lines (TPC-1, K1, NIM-1, B-CPAP and HTC-C3) were seeded at 12.5!10 4 cells/cm 2 , cultured for 18 h, and exposed to drugs for the times indicated. Cell pellets were solubilized as previously described (Caccia et al. 2010) (link). Normal, papillary and anaplastic frozen tissue samples (w50 mg) were homogenized and solubilized as previously described. Protein concentrations were determined with the bicinchoninic acid (BCA) assay (Bio-Rad Laboratories). Protein separation by electrophoresis (SDS-PAGE) and electroblotting were performed as previously described (Gorla et al. 2009 (link)) western blot quantifications were performed using Quantity one software (Bio-Rad).
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