The SMG exposed and in parallel, the ground control cells from the central 3 mm area of the flask were scratched and transferred into a 15 mL tube with 5 mL ice-cold PBS. After centrifugation at 1500 rpm for 5 min protein was extracted by bath sonication using RIPA lysis buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS; Sigma). Isolated protein was then quantified by using a standard BCA reagent kit (Thermo Fisher Scientific). Then protein samples were separated by 20% SDS-polyacrylamide gel electrophoresis. After electrophoresis proteins were transferred into the nitrocellulose membrane by using blot, a dry blot instrument (Thermo Fisher Scientific) and the transfer buffer. After the transfer, membranes were treated with 5% non-fat milk in TBS buffer for 1 h at room temperature. Membranes were then treated overnight with the primary antibodies in a dilution of 1:200 to label the targeted protein. Membranes were then washed 3 times with TBST. The dyes IRDye 680CW and 800CW (Thermo Fisher Scientific) were used. Membranes were then incubated with the secondary antibody (1:12000 dilution) for 1 h. Then membranes were washed again for 3 times and then protein bands were detected by using the Odyssey Infrared Imaging System (LI-COR).
Bca reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BCA reagent kit is a colorimetric assay used for the quantitative determination of total protein concentration. The kit contains a bicinchoninic acid (BCA) solution and a copper(II) sulfate solution, which when combined with a protein sample, produce a purple-colored complex that absorbs light at 562 nm. The intensity of the color is proportional to the protein concentration, allowing for accurate measurement of protein levels in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Total protein extraction kit, by Applygen (1 mentions)
Chemiluminescence method, by Merck Group (1 mentions)
Hif 2α, by Cell Signaling Technology (1 mentions)
Radio immunoprecipitation assay lysis buffer ripa, by Solarbio (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
β actin, by Merck Group (1 mentions)
Chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
β endorphin elisa kit, by MyBioSource (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto plus chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Novex tris glycine gel, by Thermo Fisher Scientific (1 mentions)
C ebpβ, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
17 protocols using bca reagent kit
1
Western Blot Protein Analysis Protocol
2
Rat BMSC Exosome Isolation Protocol
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Rat bone marrow mesenchymal stromal cells (BMSCs) were harvested and cultured with DMEM medium containing 10% FBS as previously described [33 (link)]. When the cell confluency reached 30–50%, the original medium was discarded and DMEM basic medium without FBS was added after the cells were washed with PBS three times. After 48–72 h, the cell culture supernatant was collected and underwent ultracentrifugation to collect exosomes. The detailed steps are as follows: centrifuge at 300 g for 10 min, take the supernatant, and remove the pellets to remove the cells; centrifuge at 2000 g for 10 min, take the supernatant, and discard precipitate to remove dead cells; centrifuge at 10,000 g for 30 min to remove the cell fragments in the sediment; centrifuge at 100,000 g for 70 min by ultracentrifugation; resuspend pellets at the bottom of the centrifuge tubes with a small amount of PBS. The concentration was quantified with a BCA reagent kit (#23225, Thermo Fisher Scientific, USA).
3
Protein Extraction and Western Blot Analysis
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The total proteins from cell samples or hearts were extracted and homogenized by using the Total Protein Extraction Kit (P1250, Applygen Technologies, Beijing, China) as per the manufacturer's instruction. After centrifuging at 2000 × g for 5 min, the clarified supernatant was separated from the sample. Then we used BCA reagent kit (23227, Thermo, MA, USA) to quantify protein concentration according to the instructions. Protein was separated on 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidine fluoride (PVDF) membrane. Next, the PVDF membrane was blocked in 10% no-fat milk for 2 h and then incubated with primary antibodies of DNP, O-GlcNAc, OGT, OGA, GFAT and COX10, respectively, at 4°C overnight, followed by incubating with horseradish peroxidase-conjugated goat secondary antibody (1:1000, in 5% no-fat milk) at room temperature for 2 h. The same membrane was probed with anti-GAPDH or anti-Porin to control lane loading.
4
Protein Extraction and Western Blot
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Extracted proteins from tissues and cells using Radio Immunoprecipitation Assay lysis buffer (RIPA, R0010) from Solarbio Life Sciences (Beijing, China). The BCA reagent kit from Thermo Scientific (23225, Waltham, USA) was used to detect protein concentration. Protein lysates were loaded on a 10% tris-glycine gel and transferred to a 0.22 μm polyvinylidene difluoride membrane. The primary antibodies (Supplementary Table 3
5
Western Blot Protein Quantification
6
Quantifying β-endorphin Levels in hSOD1G93A Mice
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The hypothalamus of hSOD1G93A mice treated with vehicle or TRAM‐34 + rimonabant was disrupted with a homogenizer and analysed for β‐endorphin content using a commercially available β‐endorphin elisa kit, in accordance to manufacturer's instructions (MyBioSource). Briefly, after two freeze–thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 min at 5000× g, 2–8°C. The supernatant was removed and assayed immediately. BCA Reagent Kit was used to measure protein concentration in samples (Thermo Scientific) and β‐endorphin content was calculated as pg·ml−1.
7
Protein Extraction and Quantification
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Total proteins from tissues or cells were extracted by cell lysis buffer with additional protease inhibitor cocktail (Calbiochem) and PMSF (Millipore). Protein concentration was measured by the BCA reagent kit (Thermo). Proteins were subjected to immunoblot assay as previously reported.27
8
Western Blot Analysis of Protein Signaling
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Total protein was extracted from cells using RIPA buffer (Boston Bioproducts) containing 1× protease/phosphatase inhibitors cocktails (ThermoFisher) and quantified using the BCA reagent kit (ThermoFisher). Thirty μg of protein were electrophoresed in 10% Novex™ Tris-Glycine Gels (ThermoFisher), transferred to nitrocellulose membranes (Bio-Rad) followed by blocking in 5% non-fat milk in TBS/0.1% TWEEN for 1 hour. Membranes were then probed overnight with the primary antibody in TBS/0.1% TWEEN. The primary antibodies used were: Phospho-p65 and total p65, phospho-STAT3 and total STAT3, C/EBP-β, and β-Actin as a loading control (Cell Signaling). Corresponding secondary antibody-HRP conjugate were used (Cell Signaling). Proteins were visualized using SuperSignal™ West Pico PLUS Chemiluminescent Substrate or SuperSignal™ West Femto PLUS Chemiluminescent Substrate (ThermoFisher).
9
Protein Quantification of Extracellular Vesicles
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A micro bicinchoninic acid (BCA) reagent kit (ThermoFisher Scientific, Waltham, MA, USA), with a bovine serum albumin (BSA) standard curve (0–200 μg/mL), was used to determine total protein in samples, as per the manufacturer’s recommendations. EVs were lysed by addition of RIPA buffer 1:1 and incubation on ice for 25 min, followed by centrifugation at 10,000× g for 10 min at 4 °C. Working reagent (WR) was prepared using MA:MB:MC at a 25:24:1 ratio, respectively. EV containing samples were diluted between 1:20–1:100 in 0.2-µm-filtered PBS; 150 μL of the WR was mixed with either 150 μL of the BSA standard or 150 μL of sample in duplicate using a 96-well plate. The plate was covered and incubated at 37 °C for 2 h. Absorbance was measured at 562 nm using a SpectraMax plate reader (Molecular Devices, San Jose, CA, USA).
10
Protein Extraction and Western Blot Analysis
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Cells were trypsinized and centrifuged at 370 g for 2 min. Cell pellet was resuspended in RIPA buffer with protease inhibitor cocktail (Sigma cat: S8830) and left to stand at 4° C for 30 min. The homogenate was centrifuged at 4° C for 15 min at 13200 g and supernatant was collected. Protein content was measured with the BCA reagent kit (ThermoScientific cat: 23225). About 100 μg of proteins were separated on 10% polyacrylamide gel (0.375 M Tris, pH 8.8, 0.1% SDS, 10% acrylamide, 0.03% ammonium persulfate (APS), and 0.06% N,N,N′,N′- tetramethylethyilenediamine (TEMED)), and transferred to a hydrophobic polyvinylidene difluoride (PVDF) membrane. The nonspecific sites of the membrane were blocked with 5% fat-free milk in 0.1% PBS-Tween for 1 h at room temperature. PVDF membrane was incubated with the primary antibody overnight at 4° C. Membrane was washed three times with 5% fat-free milk in 0.1% PBS-Tween for 1 h at room temperature and the membrane was incubated with the secondary antibody for 1 hour at room temperature. The samples were visualized with the chemiluminescent substrate ECL (GE Healthcare).
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