Protein concentrations were measured using a BCA protein kit (Bio-RAD). SOD activity in myocytes was determined using a Superoxide Dismutase Assay Kit (Trevigen, USA), according to manufacturer's instructions. The reaction involved xanthine and xanthine oxidase converting nitroblue tetrazolium (NBT) to NBT-diformazan, generating superoxide radicals, followed by SOD forming hydrogen peroxide (H2O2) from superoxide radicals. Total SOD activity was determined by the extent of reduction in the appearance of NBT-diformazan.
Bca protein kit
Manufactured by Bio-Rad
Sourced in United States
The BCA protein kit is a colorimetric assay used for the quantitative determination of total protein concentration. It employs bicinchoninic acid (BCA) as the detection reagent, which forms a purple-colored complex with copper ions in an alkaline environment. The intensity of the color is proportional to the protein concentration, enabling the measurement of protein levels in samples.
Automatically generated - may contain errors
Lab products found in correlation
Ecl system, by Merck Group (3 mentions)
Superoxide dismutase assay kit, by Bio-Techne (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by GenDEPOT (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Semi dry transfer system, by Bio-Rad (2 mentions)
Proteinase inhibitors cocktail, by Roche (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
α sma, by Abcam (1 mentions)
Horseradish peroxidase coupled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ecl plus western blotting detection reagent, by Cytiva (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protease inhibitor, by Solarbio (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Sangon (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Appropriate horseradish peroxidase conjugated secondary antibodies, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
13 protocols using bca protein kit
1
Measuring Superoxide Dismutase Activity
2
Protein Extraction and Western Blot
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Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM Na3VO4) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride; 100 μg/mL leupeptin; 10 μg/mL pepstatin, 1 μg/mL aprotinin; 2 mM EDTA) and a phosphatase inhibitor cocktail (GenDEPOT, Baker, TX, USA). After incubation for 20 min at 4 °C, the lysates were centrifuged at 13,000 rpm for 20 min at 4 °C, and the supernatants were collected for western blotting. Protein concentration was measured using a BCA protein kit (Bio-Rad, Hercules, USA). The lysates were subjected to SDS-PAGE gel electrophoresis, transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and incubated with 5% skimmed milk for 1 h at room temperature. The membranes were then probed with primary antibodies against α-SMA (Abcam, Cambridge, MA, USA), FPR2 (Novus Biological, Littleton, CO, USA), and actin (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with horseradish peroxidase-coupled secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). Proteins were detected using ECL Plus western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA).
3
Protein Extraction and Western Blot Analysis
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Total protein extraction was carried out by using the RIPA lysis buffer (Sangon Biotech, Shanghai, China) with protease inhibitor (Solarbio), according to the manufactory's description. After quantification with a BCA Protein Kit (Bio‐Rad Laboratories), 30 μg proteins of each sample were separated by 10% polyacrylamide gels and were transferred to polyvinylidene difluoride membranes (Millipore). The membranes were then incubated with 5% non‐fat milk and probed with the primary antibodies overnight at 4°C, including NUSAP1 (1:1000 dilution; No. ab137230, Cambridge, MA, USA), Bcl2 (1:1000 dilution; No. #15071, Cell Signaling Technology), Bax (1:1000 dilution; No. #2772, Cell Signaling Technology), E‐cadherin (1:1000 dilution; No. #3195, Cell Signaling Technology), N‐cadherin (1:1000 dilution; No. #4061, Cell Signaling Technology), and GAPDH (1:2000 dilution; No. #2118, Cell Signaling Technology). After the incubation with the corresponding second antibodies (Santa Cruz Biotechnology), the protein expression levels were determined on a Western blotting imaging and quantitative system (Bio‐Rad). Protein quantification was performed using the ImageJ software (National Institutes of Health) after background subtraction, with GAPDH level as an internal reference.
4
Western Blot Protein Analysis Protocol
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Podocytes were washed with cold PBS and then lysed with 150 μl of RIPA buffer containing proteinase inhibitors cocktail (Roche) and phosphatase inhibitors. The lysates were incubated on ice and then centrifuged 12,000 g for 15 min at 4°C. The supernatants were transferred to fresh tubes and then subjected to protein concentration measurement with BCA protein kit (Bio-Rad). After mixed with loading buffer, the samples were boiled at 98°C for 5 min. 10 or 8% SDS-PAGE was used to fractionate the samples, and semi-dry transfer system (Bio-rad) was used to transfer the protein from the gel to PVDF membrane. The blot was blocked with 5% milk in TBST solution (20 mM Tris-HCl, PH 7.14, 150 mM NaCl, 0.1% Tween-20) for 60 min at room temperature, and then incubated with an antibody overnight at 4°C. After washed with TBST for 3 times, the blot was incubated with HRP-labeled secondary antibody for 1 h at room temperature. After washed, ECL system (Millipore) was used to detect the protein. Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche) and a phosphatase inhibitor. The blot was incubated with a primary antibody of interest.
5
Western Blot Analysis of Protein Expression
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Cells were lysed with RIPA buffer containing protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor. Protein concentrations of the supernatant samples were measured with BCA protein kit (Bio-Rad). The samples were mixed with loading buffer and boiled for 5 min. Ten percent of SDS-PAGE was used for electrophoresis of the samples, and the proteins were then transferred to membrane. The membrane was incubated with blocking solution containing 5% milk in TBST solution (20 mM Tris-HCl, PH 7.14, 150 mM NaCl, 0.1% Tween-20) at room temperature for 1 h, and then incubated with primary antibodies respectively, at 4°C overnight. The membrane was washed using TBST for 5 min (3X), and then incubated with HRP-labeled secondary antibody at room temperature for 1 h. After washed, the ECL system (Millipore) was used to detect the proteins. AlphaView (ProteinSimple, USA) was used to quantify band intensities on the blots.
6
Western Blot Analysis of BECN1 Protein
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The tissues and cells were lysed in Radio-Immunoprecipitation buffer (Beyotime), and quantified by bicinchoninic acid (BCA) protein kit (Bio-Rad, Hercules, CA, USA). Then, an equal amount of proteins was separated by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred onto the polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% non-fat milk, the membranes were incubated with anti-BECN1 (1:1000 dilution; Cell Signaling Technology, Cambridge, MA, USA) or β-actin (1:2000 dilution; Cell Signaling Technology) overnight at 4 °C. Appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution; Cell Signaling Technology) were applied to detect labeled proteins. The protein bands were visualized with SuperSignal Ultra Chemiluminescent Substrate (Pierce, Rockford, IL, USA) on imaging system (Protein Simple, Santa Clara, CA, USA).
7
Protein Expression Analysis in Colon Tissue
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Total proteins from colon tissue and IECs were extracted with RIPA buffer. The lysates were centrifuged at 12,000 × g for 15 min, and the protein concentration was detected with a BCA protein kit (Bio-Rad, USA). Next, the proteins were separated by SDS-PAGE and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk at room temperature for 2 h and incubated with the primary antibodies against anti-occludin (ab216327, abcam, UK), anti-ZO-1(ab96587, abcam), anti-DNMT3a (ab188470, abcam), anti-TNFSF13 (ab108206, abcam), anti-TCAI (ab239370, abcam), and anti-β-actin (ab8226, abcam) at 4°C overnight. Then, the PVDF membrane was incubated with the HRP-conjugated secondary antibody. Finally, the bands were visualized by enhanced chemiluminescence (ECL) kit and gel imaging system.
8
Purification and Characterization of Recombinant VCAM-1 Proteins
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Recombinant VCAM-1 protein with the His tag was purified using His GraviTrap (GE Biosciences, Sweden) and recombinant VCAM-1 protein with the GST tag was purified using Gluthathione-Sepharose 4B (GE Healthcare, USA), following kit protocols for both purifications. The recombinant proteins His-VCAM-1 and GST-VCAM-1 were analysed by 12% SDS-PAGE and Western blot using monoclonal antibodies against the His and GST tags (Sigma, USA) respectively. The protein concentrations of both purified recombinant proteins were determined using a BCA protein kit (Bio-Rad, USA).
9
Western Blot Analysis of Podocyte Proteins
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After the treatment, podocytes were washed with cold PBS and then lysed with 150 μl of RIPA buffer containing proteinase inhibitors cocktail (Roche) and phosphatase inhibitors. The lysates were incubated on ice and then centrifuged 12,000 g for 15 min at 4 °C. The supernatants were transferred to fresh tubes and then subjected to protein concentration measurement with BCA protein kit (Bio-Rad). After mixed with loading buffer, the samples were boiled at 98 °C for 5 min. 10% or 8% SDS-PAGE was used to fractionate the samples, and semi-dry transfer system (Bio-rad) was used to transfer the protein from the gel to PVDF membrane. The blot was blocked with 5% milk in TBST solution (20 mM Tris-HCl, PH 7.14, 150 mM NaCl, 0.1% Tween-20) for 60 min at room temperature, and then incubated with antibody overnight at 4 °C. After washed with TBST for 3 times, the blot was incubated with HRP-labeled secondary antibody for 1 h at room temperature. After washed, ECL system (Millipore) was used to detect the protein.
10
Measuring Cellular Antioxidant Enzyme SOD
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Protein concentrations were measured by BCA protein kit (Bio-RAD, Hercules, CA, USA). SOD activities in CSPCs were determined by the Superoxide Dismutase Assay Kit (Trevigen, Gaithersburg, MD, USA), and followed the manufacturer’s instructions. The reaction involved xanthine and xanthine oxidase converting nitroblue tetrazolium (NBT) to NBT-diformazan, generating superoxide radicals, and then SOD formed H2O2 from superoxide radicals. Total SOD activity was determined by the extent of reduction in the appearance of NBT-diformazan. Activity was evaluated using 50–500 μg of total cellular protein with 5 mM sodium cyanide (NaCN) to inhibit SOD1 activity. Total SOD activity (reactions not containing NaCN) and SOD2 activity (reactions containing 5 mM NaCN) in U/mg protein were finally calculated in each experimental sample. SOD1 activity was measured by subtracting the SOD2 activity from the total SOD activity.
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