Lymphocytes were seeded in six-well plates at a concentration of 106cells/well, incubated overnight and treated with chemicals for 24 h. Then cells were lysed and total protein levels were determined using the Bio-Rad Bradford assay kit (Bio-Rad, UK). The cell lysates were separated using protein electrophoresis and blotted on nitrocellulose membrane (Abcam, UK). The membranes were blocked overnight in 5% bovine serum albumin (BSA) diluted in Tris-buffered saline supplemented with 0.1% Tween 20 at 4 °C. The membranes were then incubated with primary and secondary antibody dilutions, overnight at cold and for 1 h at room temperature, respectively.
Bradford assay kit
Manufactured by Bio-Rad
Sourced in United States, Germany, United Kingdom, Italy
The Bradford assay kit is a colorimetric assay used for the quantitative determination of protein concentration. The kit utilizes the Bradford dye-binding method, where the dye binds to protein, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl reagent, by Thermo Fisher Scientific (16 mentions)
Protease inhibitor cocktail, by Roche (11 mentions)
Phosphatase inhibitor cocktail, by Merck Group (10 mentions)
Protease inhibitor cocktail, by Merck Group (10 mentions)
Complete protease inhibitor cocktail, by Roche (8 mentions)
Peroxidase conjugated goat anti mouse igg or goat anti rabbit igg, by Jackson ImmunoResearch (7 mentions)
Wet transfer apparatus, by Bio-Rad (6 mentions)
Pvdf membrane, by Cytiva (6 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (6 mentions)
Trypsin, by Promega (6 mentions)
Protease inhibitor, by Roche (6 mentions)
Nitrocellulose membrane, by GE Healthcare (6 mentions)
Ecl kit, by Cytiva (5 mentions)
Pro pre protein extraction solution, by iNtRON Biotechnology (5 mentions)
β actin, by Merck Group (5 mentions)
Tissuelyser lt, by Qiagen (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
Nitrocellulose membrane, by Cytiva (5 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by GE Healthcare (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
296 protocols using bradford assay kit
1
Protein Expression Analysis in Lymphocytes
2
Protein Sample Preparation for SDS-PAGE and 2DE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For SDS-PAGE, protein precipitates were dissolved in a SDS-containing buffer (0.5% SDS, 50mM Tris-HCl, pH 6.8, and 20 mM DTT). Protein concentration was determined using the Bio-Rad Bradford assay kit (Bio-Rad, Hercules, CA) [21 (link)], but performed on a micro scale, i.e., 10 μl of standard or sample solution was mixed with 1.0 ml of diluted dye solution. In this way, the final concentration of SDS in the mixture was 0.005%, which was compatible with the Bradford assay. Prior to SDS-PAGE, protein extracts were mixed with appropriate volume 4 x SDS sample buffer [22 (link)]. For 2DE, protein precipitates were dissolved in the 2DE rehydration solution without IPG buffer to avoid its interference as we described before [23 (link)], and protein concentrations were determined by the Bradford Assay. Subsequently, the IPG buffer was supplemented into protein samples to a concentration of 0.5%.
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein from the transfected cells was obtained using a lysis buffer containing 1% phosphatase inhibitor cocktail II, 0.2 mM PMSF, and 2% Trixon-X (Sigma-Aldrich, St. Louis, MO, USA), and protein concentration was determined using a Bradford assay kit (Bio-Rad, Hercules, CA, USA). Protein samples (20 µg) were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham, Braunschweig, Germany), which were blocked with 5% skim milk in TBS-Tween 20 (TTBS) for 1 h and then incubated overnight with the indicated primary antibodies (Table S3 ) at 4 °C, followed by incubation with secondary antibodies (Table S3 ) for 1 h at room temperature. Blots were developed using a Femto reagent (Thermo Fisher Scientific, Waltham, MA, USA) with a Fusion Solo Chemiluminescence Imaging System (Vilber Lourmat, Marne-la-Vallée, France), and their densities were analyzed with Evolution Capt software (Vilber Lourmat). β-Actin protein levels were used for normalization.
4
Sildenafil-Induced Protein Profiling in Mature Forebrain Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At day 40, mature forebrain neurons treated with sildenafil were harvested, washed with cold PBS, and then lysed with N-PERtrademark Neuronal Protein Extraction Reagent (Cat# 87792, Thermo Scientific) adding 1% Protease/Phosphatase Inhibitor (Cat# PI78442, Thermo Scientific). Total protein concentration was measured using the Bradford assay kit (Bio-Rad, USA) according to the manufacturer’s manual. Samples were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a polyvinylidene difluoride (PVDF; EMD Millipore, Darmstadt, Germany) membrane. After transferring, the membranes were probed with specific primary antibodies (1 : 1000) at 4°C overnight. Specific protein bands were detected using a chemiluminescence reagent after hybridization with a horseradish peroxidase (HRP)-conjugated secondary antibody (1 : 8000). Band intensities were normalized with GAPDH and compared by performing densitometry analysis (ImageJ). For pTau231/total tau measurement, lysate was run on a Phospho (Thr231)/Total Tau Kit (K15121D, MSD, USA) following manufacturers protocol with minor modifications.
5
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed in ice-cold PBS with Ca2+/Mg2+ and lysed in ice-cold RIPA lysis buffer containing Phosphatase Inhibitor Cocktails 2 and 3 and Protease Inhibitor Cocktail (Sigma-Aldrich). After centrifugation at 13′000 g for 10 min at 4°C, supernatants were collected and protein concentration was measured using Bradford assay kit (Bio-Rad) or BCA assay (Fisher Scientific) for Rho-pulldown samples. The samples were mixed with NuPAGE LDS sample buffer and reducing agent (Invitrogen) and heated at 70°C for 10 min 10 μg total protein was loaded onto a 4–12% NuPAGE BT gradient gel (Invitrogen), resolved by SDS-PAGE, and transferred to a 0.45 μm polyvinylidene fluoride membrane in NuPAGE Transfer Buffer at 20V for 1h using a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked for 30 min with 5% BSA in PBS buffer containing 0.1% Tween 20 (Sigma-Aldrich), incubated with primary antibodies in PBS/Tween 20 overnight at 4°C, washed in PBS/Tween 20, incubated with appropriate HRP-conjugated secondary antibodies for 1 h, and developed with Super-Signal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins and nuclear extracts were isolated by the Nuclear and Cytoplasmic Protein Extraction Kit according to the manufacturer’s instruction. Cells were seeded in 60 mm dishes with a density of approximately 3 × 105 cells/well for western blot tests. The protein concentrations were determined using the Bradford Assay kit (Bio-Rad) and the cell lysates were boiled with 5 × SDS gel-loading dye for 10 min at 100°C. The samples (20 μg/lane) were electrophoresed on 12% SDS-PAGE gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA). After blocked with 5% non-fat milk in TBST buffer (0.1% Tween-20) for 1 h at room temperature, the membranes were incubated with the primary antibodies of interest at 4°C overnight. After washed the membranes three times with TBST, the membranes were exposed to horseradish peroxidase conjugated secondary antibodies for 2 h at room temperature. After washed the membranes three times with TBST, proteins bands were visualized by an ECL detection kit following the manufacturer’s instructions.
7
Protein Extraction from Tissues and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extracts were prepared from quadriceps and brain tissue as detailed elsewhere [18 ]. Briefly, snap-frozen tissues were homogenized on ice, in RIPA lysis buffer using a loose pestle. Protein lysate was then collected via microcentrifugation. For protein extracts from cells, cold Tris lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1% Triton-X 100, Protease and Phosphatase inhibitor cocktail) was added directly to the culture dish, and cells were collected by scraping the dish. Cells in lysis buffer were transferred to an Eppendorf tube and incubated for 30 min on ice with periodic agitation. Samples were then microcentrifuged for 30 min at 4 °C, after which the supernatant was transferred to a fresh tube. All extracts were stored at − 70 °C until analysis. Protein concentration was quantified using the Bradford assay kit (Bio-Rad), prior to western blot analysis.
8
Anti-Inflammatory Drug Screening in LPS-Stimulated RAW264.7 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RAW264.7 cells (1 × 106 cells/mL) were incubated for 12 h in 6-well plates, and then pre-treated with different concentrations of tested compounds in the presence of LPS (1 μg/mL). After incubation for 18 h, cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed on ice by using lysis buffer (C-3228, Sigma) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma) for 10 min. Then, lysates were centrifuged at 12,000× g for 10 min at 4 °C and the cytoplasmic proteins were collected. To detect nuclear protein, cells (1 × 106 cells/ mL) were extracted with NE-PER nuclear and cytoplasmic extraction reagent kits (Pierce, Rockford, IL, USA) to analyze the nuclear translocation of NF-κB subunit p65. All protein concentrations were determined by a Bradford assay kit (Bio-Rad, Hercules, CA, USA).
9
BALF Harvesting and Differential Cell Counting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain BALF, an 18-gauge catheter was inserted into the trachea and perfused twice with 800 µL of autoclaved PBS through the tracheal cannula as described previously [14 (link)]. Collected BALF was then centrifuged at 400 g for 10 min at 4°C, supernatant was transferred to fresh tubes, and pellets were resuspended in PBS. Total protein content in BALF was determined using a Bradford assay kit (Bio-Rad, Hercules, CA) according to the manufacturer's protocol. In brief, 10 µL of BALF and 200 µL of Bradford agent were added to a 96-well plate and incubated for 5 min at room temperature. The absorbance was measured at 595 nm using a spectrophotometer (Molecular Devices, Sunnyvale, CA). To count differential cells, pellets were resuspended in PBS, fixed onto slides, and stained using a Diff-Quik staining kit (Sysmex Co., Kobe, Japan). Total cell numbers in BALF were determined using a hemacytometer.
10
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in PRO-PRE-Protein Extraction Solution (Intron Biotechnology, Seongnam, Korea). Protein concentrations were determined using a Bradford assay kit (BIO-RAD, Hercules, CA, USA). Cell lysates (50 μg of total protein) were separated in 8% acrylamide gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond-ECL nitrocellulose filter paper (Amersham Biosciences, Buckinghamshire, UK). Membranes were blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. Protein bands were probed with VEGFR2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a 1:500 dilution; phospho-VEGFR2 (Tyr951, Tyr1175), total-ERK (Thr202/Tyr204), phosphor-ERK (Thr202/Tyr204), total-AKT, phospho-AKT total-p38, phospho-p38 (Cell Signaling, USA) at 1:1000 dilutions; β-actin antibody at a 1:4000 dilution (Santa Cruz Biotechnology, Santa Cruz, USA) and then labeled with horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Piscataway, USA). Bands were visualized by enhanced chemiluminescence using an ECL kit (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer’s protocol, as described previously23 (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!