Cell lines were plated in 6-well plates with a density of 5 × 105 cells per plate. Intracellular ATP was measured using a luciferin/luciferase-based assay (Adenosine 5'-triphosphate (ATP) Bioluminescent Assay Kit, Sigma-Aldrich) following manufacturer's guidelines. A standard curve was generated rather than known concentrations of ATP and used to calculate sample ATP concentrations. Protein concentration was determined using Bradford protein assay reagents (Bio-Rad). The content of ATP was normalized for protein content and expressed as percentage of control.
Bradford protein assay reagent
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, China, Italy
The Bradford protein assay reagent is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the binding of the dye Coomassie Brilliant Blue G-250 to proteins, which results in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Protease inhibitor cocktail, by Roche (11 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Phosphatase inhibitor cocktail, by Merck Group (8 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (6 mentions)
Supersignal west pico, by Thermo Fisher Scientific (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ripa buffer, by Merck Group (5 mentions)
Wet transfer apparatus, by Bio-Rad (5 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
3h 2 deoxy d glucose, by PerkinElmer (5 mentions)
Humulin r insulin, by Eli Lilly (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Enhanced chemiluminescence reagent, by Cytiva (4 mentions)
Enhanced chemiluminescence, by Cytiva (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Chemidoc imaging system, by Bio-Rad (4 mentions)
Protease inhibitor, by Merck Group (4 mentions)
136 protocols using bradford protein assay reagent
1
Intracellular ATP Quantification Protocol
2
Western Blot Analysis of Liver Proteins
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Liver tissues collected from WT and CF rabbits were homogenized using an electric homogenizer followed by centrifugation at 4°C. The suspension was frozen at −80°C until analysis. Total protein concentration was determined by the Bradford Protein Assay Reagents (Bio-Rad, USA). 40 µg total protein per sample was used for Western blotting. Blotted membranes were incubated overnight at 4°C with the appropriate primary antibodies (Table S2 ). After washing, the membranes were incubated with the secondary antibody for 2 hours. After three times of wash, the reactive bands were visualized by incubation with enhanced chemiluminescence substrates and exposure to an X-ray film. The signal intensities were determined by Quantity One (Bio-Rad Life Science, Hercules, California, CA, USA).
3
Homocysteine-Induced Neuronal Damage
4
Protein Extraction Reagents and Assay
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Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), sodium dodecyl sulfate (SDS), bovine serum albumin, dimethyl sulfoxide (DMSO), and ethylenediaminetetraacetic acid (EDTA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Skim milk was purchased from BD Difco Laboratories, Inc., a subsidiary of Becton, Dickinson, and Company (Sparks, MD, USA). Bradford protein assay reagents and 30% bis-acrylamide solution were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). All other chemicals and organic solvents used in this study were of the highest grade from commercial sources.
5
Western Blot Protein Analysis Protocol
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Cells were lysed with RIPA lysis buffer (GenDEPOT). After protein quantification using Bradford protein assay reagents (Bio-Rad, Hercules, CA, USA), equal amounts of protein were loaded into the well of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and fractionated. After transfer onto a nitrocellulose membrane (Bio-Rad), membranes were blocked with 5% bovine serum albumin (BSA) in PBS + 0.1% Tween 20 (0.1% PBST) for 1 h at RT. After incubation with primary antibodies at 4 °C overnight, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. The blot was developed by ECL chemiluminescence detection system (Abfrontier, Seoul, Republic of Korea).
6
Comprehensive Antibody Profiling Protocol
7
4-HNE Protein Adducts Analysis
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Protein from frozen striatum and midbrain was extracted with 1 × radioimmunoprecipitation assay buffer (CST, Beverly, MA, USA) with 1 × Calbiochem Protease Inhibitor Cocktail Set I and 1 × Halt* Phosphatase Inhibitor Cocktail (Thermo Scientific). A Tissue Lyser LT (Invitrogen, Pleasanton, CA, USA) with a 5 mm steel bead was used to homogenize tissues at 50 Hz. Tissue lysate was then centrifuged at 13,000 rpm for 15 min at 4°C. Supernatant was saved in a –80°C freezer. Bradford protein assay reagents (Bio-Rad, Irvine, CA, USA) were used to determine protein concentration. Protein (40 μg/lane) was then separated on a 4–12% Criterion sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (Bio-Rad) and transferred on to nitrocellulose membrane. Proteins were detected with antibodies directed specifically to 4-HNE protein adducts (R&D, Minneapolis, MN, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST), respectively, in the midbrain and striatum of 6- and 18-month-old mice. An appropriate secondary antibody from LiCor (IR800 or IR680) was used corresponding to each primary antibody. Immunoreactive bands were imaged and quantified using Odyssey software (LiCor, Lincoln, NE, USA).
8
Western Blot Analysis of Protein Levels
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The liver tissue samples and macrophage cells were lysed in radioimmunoprecipitation assay buffer (Millipore, Bedford, MA, USA) for total cell protein or in NE-PER extraction reagent (Thermo) for cytosolic and nuclear proteins. Concentrations of total protein were measured by Bradford protein assay reagents (Bio-Rad, Hercules, CA, USA). Equal amount of proteins was separated and then blotted in accordance with a previously described method [20 (link)]. Proteins on the membrane were blocked and then incubated with various primary antibodies followed by secondary antibodies (Table 2 ). Immunoreactive bands of target protein were detected using enhanced chemiluminescence solution (Bio-Rad). Each detected protein band was normalized by internal control proteins and was quantified using ImageJ software (version 1.53k).
9
Quantification of Intracellular and Extracellular PpIX
10
Antibody-Based Detection of MK5 and ERK3/4
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Medium 199 and fungizone were from Sigma-Aldrich, fetal bovine serum (FBS) and trypsin were from Gibco Laboratories (Life Technologies, Inc.), and penicillin-streptomycin solution was from Multicell. All culture plates were from Sarstedt, Inc. PCR Primers were from Invitrogen. SDSpolyacrylamide gel electrophoresis reagents, nitrocellulose, and Bradford protein assay reagents were from Bio-Rad Laboratories. Antibodies against MK5 were from Santa Cruz Biotechnology (PRAK A-7) and Cell Signaling Technology (MAPKAPK-5 #D70A10). Antibodies against phosphothreonine182-MK5 were from Abcam (pT182-MK5, ab138668). ERK3 antibodies were from Santa Cruz Biotechnology (ERK3 D23) and Thermo Fisher Scientific (ERK3 5E1).
Antibodies to ERK4 were from Santa Cruz Biotechnology (ERK4 N20). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.
Proximity Ligation Assay kits from Sigma-Aldrich. Lambda protein phosphatase and alkaline phosphatase were from Calbiochem (#539514-20KV) and Sigma-Aldrich (#P6774-2KU), respectively. Collagen-coated hydrogel-bound (8 kPa) polystyrene 35mm (PS35-COL-8) and 6well (SW6-COL-8) plates were from Matrigen. Phos-tag acrylamide was from Wako Chemicals (#AAL-107). All other reagents were of analytical grade or best grade available. Milli Q-grade (>18.2 MΩ/cm) water was used throughout these studies.
Antibodies to ERK4 were from Santa Cruz Biotechnology (ERK4 N20). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories.
Proximity Ligation Assay kits from Sigma-Aldrich. Lambda protein phosphatase and alkaline phosphatase were from Calbiochem (#539514-20KV) and Sigma-Aldrich (#P6774-2KU), respectively. Collagen-coated hydrogel-bound (8 kPa) polystyrene 35mm (PS35-COL-8) and 6well (SW6-COL-8) plates were from Matrigen. Phos-tag acrylamide was from Wako Chemicals (#AAL-107). All other reagents were of analytical grade or best grade available. Milli Q-grade (>18.2 MΩ/cm) water was used throughout these studies.
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