Samples of tibialis anterior muscle biopsies were lysed in RIPA buffer (150 mM NaCl, 25 mM Tris-Cl, pH 7,4, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate) and supplemented with protease and phosphatase inhibitors. Following the protein extraction protocol, protein concentration was measured using the bicinchoninic acid (BCA) method, according to the manufacturer’s instructions (Pierce™ BCA Protein Assay Kit, Sigma Aldrich, Poole, UK). The proteins (40 μg) were separated by electrophoresis, transferred to nitrocellulose membranes, and stained proteins with Ponceau S. After that, the membranes were incubated with primary antibodies against FoxO1 (2880) (Cell Signalling, Danvers, MA, USA), MuRF1(SC32920) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 20S (PW8195) (Enzo Life Sciences, Farmingdale, NY, USA), and GAPDH (SC47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a loading control. Later, the membranes were probed with secondary antibodies conjugated with peroxidase (secondary antibodies goat antirabbit (7074) and horse antimouse (7076) (Cell Signalling)), and bands were visualised using a chemiluminescent reagent (Thermo Fisher Scientific, Waltham, MA, USA). The membrane images were captured using an imaging system (Amersham Imager 600, GE Healthcare), and band volume quantitation was quantified.
Pierce bca protein assay kit
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
The Pierce™ BCA Protein Assay Kit is a colorimetric detection method used to quantify the total protein concentration in a sample. The assay relies on the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the resulting purple-colored reaction is measured spectrophotometrically.
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Lab products found in correlation
Digital sonifier 450, by Emerson (4 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Meso quickplex sq 120 plate reader, by Mesoscale (2 mentions)
V plex plus mouse cytokine 19 plex elisa kit, by Mesoscale (2 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Spectramax id5, by Molecular Devices (2 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Gapdh sc 47724, by Santa Cruz Biotechnology (1 mentions)
Foxo1 2880, by Cell Signaling Technology (1 mentions)
Glucose go assay kit, by Merck Group (1 mentions)
Glycolysis cell based assay kit, by Cayman Chemical (1 mentions)
Recombinant murine g csf, by Thermo Fisher Scientific (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
Trans blot apparatus, by Bio-Rad (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mouse anti vimentin mab, by Agilent Technologies (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Biotrace nt, by Pall Corporation (1 mentions)
20 protocols using pierce bca protein assay kit
1
Protein Expression Analysis in Tibialis Anterior
2
Profiling Colon Inflammatory Cytokines
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Colon homogenates were directly prepared from frozen tissue samples in ice-cold lysis buffer (water with 500 nM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS, 50 mM Tris HCl and 1 tablet of protease inhibitor/mL of solution) by sonication (Branson 450 digital sonifier®, 30% amplitude, 10 s) (15 mg tissue/100 μL of lysis buffer). Samples were subsequently centrifuged at 20,000 × g for 10 min. The supernatant was retrieved and kept at −80 °C until further analysis, and the pellet was discarded. The concentrations of the following colonic proinflammatory cytokines and chemokines were determined with a V-PLEX Plus Mouse Cytokine 19-Plex ELISA kit (MesoScale) following the manufacturer's instructions using a MESO QuickPlex SQ 120 plate reader (MesoScale): IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, KC/GRO, IL-10, IL-12p70, Il-15, IL-17AF IL-27p28/IL-30, IL-33, IP-10, MCP-1, MIP-1α, MIP-2 and TNF-α. The concentrations were normalized to the protein content, quantified with a Pierce™ BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, USA).
3
Glycolysis Profiling of Atg7 KO Cells
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32D CRISPR-Atg7–/– and Atg7+/+ clones were cultured in RPMI 1640 (containing 2mg/mL Glucose) with 1% FCS, 2 mM L-glutamine, 100 U/ml Pen-Strep. In order to induce neutrophil differentiation, 1x106 cells/ml were seeded in 96-well plates with medium supplemented with 100 ng/ml recombinant murine G-CSF (peprotech). At day 3, medium was supplemented with fresh 100 ng/mL G-CSF. Cells and supernatant were harvested at indicated time points and supernatants analyzed for glucose levels and L-lactate levels following the Glucose (GO) Assay Kit (GAGO-20, Sigma) and the Glycolysis Cell-Based Assay Kit (#600450, Cayman Chemicals). Cells were lysed in NP40 buffer and protein was quantified using Pierce BCA Protein Assay Kit (Sigma). Glucose consumption and L-lactate levels were normalized to protein levels for each well individually. Each assay representative of at least two independent experimental repeats.
4
Extracellular Vesicle Protein Quantification
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Protein quantification using Micro BCA protein assay (Pierce BCA Protein Assay Kit, Sigma‐Aldrich) followed the manufacturer's instructions. In short, EV samples were diluted in DI water (1:1 ratio), and 150 μl of the solution was incubated for 2 h at 35°C with an equal reagent volume. Absorbance was then measured at 562 nm using a ClarioStar plate reader (BMG Labtech, Germany).
5
Myeloperoxidase Activity Assay in Colonic Tissue
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The colonic tissue was placed in a 1.5 mL Eppendorf tube with 500 μL of HTAB buffer (0.5% HTAB in 50 mM potassium phosphate buffer (KH2PO4 50 mM), pH 6) on ice and gently homogenized (Branson 450 digital sonifier®, 30% amplitude, 10 s) while kept on ice. The homogenates were centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant (7, 14 or 21 μL) was added to 96-well plates (Nunc, Roskilde, DK) together with 200 μL of the MPO analysis solution (50 mM potassium phosphate buffer pH 6 containing 0.167 mg/mL o-dianisidine and 500 ppm hydrogen peroxide). The MPO activity in the supernatant was measured with a spectrophotometer (SpectraMax® iD5, Molecular Devices, LLC, USA) at 460 nm for 30 min. The results were expressed as MPO units per gram of tissue, and one unit of MPO activity was defined as the amount that degrades 1 mmol/min of hydrogen peroxide at 25 °C [25 (link),26 (link)]. The concentrations were normalized to the protein content, quantified with a Pierce™ BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, USA).
6
Total Protein Extraction and Western Blot Analysis
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For total protein collection, cells were lysed with RIPA-buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and a protease inhibitor mixture (2 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF, 5 mM EDTA, 1 mM EGTA, 10 mM NaF, and 1 mM Na3VO4). Lysed cells in 6-well plates were collected with cell scrappers and centrifuged at 23,500 × g for 20 min at 4 °C. Supernatants were collected and protein concentrations were analyzed using Pierce BCA protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). Cell lysates were stored at −80 °C until further use. For Western blot analysis, protein samples were separated using SDS-PAGE and transferred onto nitrocellulose membranes. These membranes were then blocked with 3% skim milk buffer for 1 h 30 min at room temperature and washed with Tris-buffered saline (TBS). Membranes were incubated with primary antibody at 4 °C overnight. After washing with TBS, membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase for 2 h at room temperature and visualized using ECL detection reagents (Waltham, MA, Thermo Scientific, USA).
7
Western Blot Analysis of EpCAM Subpopulations
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Total cell lysates were prepared for each of the EpCAM subpopulations, the four selected PMC42-LA clones, and and parental PMC42-LA cell line by lysing the cells in the presence of RIPA Buffer (10 mM Tris-HCl pH 7.6, 10 mM NaCl, 3mM MgCl2, 1% nonidet P-40, 1 X Protease Inhibitor tablet (Roche)) on ice. Next, protein levels were quantified using the Pierce™ BCA Protein Assay Kit (Sigma) and 30 μg of total protein from each sample was prepared with sample reducing buffer (2 M Urea, 2% SDS (sodium dodecyl sulfate), 0.125 M Tris HCl, 0.1M DTT (dithiothreitol) and bromophenol blue) at a ratio of 3:1 (lysate: reducing buffer) and resolved on an SDS gel with Tris/Glycine/SDS gel running buffer. The samples were subsequently transferred onto nitrocellulose membranes (BioTrace NT, Pall Life Sciences, New York, NY, USA) using a Transblot apparatus (Bio-Rad) and blocked using 1:1 Odyssey® blocking buffer (LI-COR): 1X PBS prior to probing with mouse anti-E-cadherin mAb (clone 36/e-cad, BD Biosciences), mouse anti-vimentin mAb (clone V9, Dako), and mouse Pan-actin mAb (clone ACTN05, Thermo Scientific). Membranes were then scanned on the Odyssey imaging system (Li-Cor, Lincoln, NE, USA) to obtain a visual representation of the amount of protein present in the samples.
8
Western Blot Analysis of Cellular Proteins
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Cell extracts were prepared in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and the concentration was measured using a Pierce BCA Protein Assay kit (Sigma-Aldrich; Merck KGaA) according to the manufacturer's protocol. Briefly, each sample (20 µg protein), prepared with loading buffer, was separated by SDS-PAGE on 8% gels and electrotransferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked in 5% non-fat milk for 1 h at room temperature and incubated with primary antibody at 4°C overnight. GAPDH primary antibody (cat. no. G8795; 1:5,000) was purchased from Sigma-Aldrich (Sigma-Aldrich; Merck KGaA), γ H2A histone family member X (γH2AX; cat. no. 33686; 1:1,000), poly [ADP-ribose] polymerase 1 (PARP1; cat. no. 31288; 1:1,000) and caspase3 (cat. no. 29034; 1:1,000) primary antibodies were from Signalway Antibody LLC (College Park, MD, USA). The membranes were then washed with three times with Tris-buffered saline and incubated with a horseradish peroxidase-conjugated secondary antibody (cat. no. 7074, 1:10,000; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h. Finally, the blots were visualized using enhanced chemiluminescence (EMD Millipore) and the semi-quantification of bands was performed using ImageJ (version 1.51; National Institutes of Health, Bethesda, MD, USA).
9
Western Blot Analysis of Protein Samples
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Cells were collected and lysed in RIPA-buffer and a protease inhibitor cocktail. Lysed cells were centrifuged at 21,000× g for 15 min at 4 °C. Protein concentration was measured using the Pierce BCA protein assay kit (Sigma-Aldrich, St. Louis, MO, USA) and cell lysates were stored at −80 °C until further use. For Western blot, protein samples (30 µg per treatment) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Following protein transfer, membranes were blocked with 3% non-fat milk buffer and then incubated overnight at 4 °C with primary antibodies, which were used at a dilution range of 1:1000 to 1:20,000. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000). The membranes were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific, Waltham, MA, USA). The density of the bands was determined using Image J software (National Institutes of Health, Bethesda, MD, USA), and normalized to that of the house-keeping protein, GAPDH.
10
Analyzing Canine Anal Sac Secretions
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Dogs were handled by their owners while one of the two anal sacs was emptied by gently squeezing the surrounding tissue. Secretion was collected in a 10 ml falcon tube, and immediately transported on ice to the lab. Samples were vortexed for 30 s and centrifuged for 15 min at 21,000g at 4 °C. Twenty microliter of supernatant was detained for total protein concentration measurement by using the Pierce BCA Protein Assay Kit (Sigma-Aldrich). The remaining supernatant was divided into aliquots and stored at − 80 °C until further handling.
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