Cell lysates were prepared, and protein concentration was quantified using the BCA assay kit (Beijing Dingguo Changsheng Biotechnology CO., Ltd. Beijing, China). Equal amounts of proteins were separated with 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and then transferred onto PVDF membranes (Millipore CO., Ltd. Beijing, China). The membranes were blocked in Tris-buffered saline/Tween 20 (TBS-T) containing 5% skim milk for 1 h and incubated with primary antibodies against URAT1, OAT1, and β-actin overnight at 4°C. After washing with the TBS-T buffer, the membrane was incubated with the secondary antibody. Specific bands were visualized on gel imager (Bio-rad, USA) by chemiluminescence using ECL™ Detection reagents (Abbkine Biotechnology Co., Ltd., Wuhan, China).
Bca assay kit
Manufactured by Dingguo
Sourced in China
The BCA assay kit is a colorimetric-based method used for the quantitative determination of total protein concentration in a sample. The kit utilizes the bicinchoninic acid (BCA) reaction to produce a purple-colored reaction product, which can be measured spectrophotometrically.
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Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Ecl reagent, by Merck Group (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Gel imager, by Bio-Rad (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Mammalian protein extraction reagent, by CWBIO (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Gsk 3β, by Santa Cruz Biotechnology (1 mentions)
Phospho gsk3β ser9, by Santa Cruz Biotechnology (1 mentions)
Cd133, by Santa Cruz Biotechnology (1 mentions)
Phospho akt thr308, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Nanog, by BD (1 mentions)
Ab178847, by Abcam (1 mentions)
8 protocols using bca assay kit
1
Western Blot Analysis of Renal Transporters
2
Protein Expression Analysis in H9c2 Cells
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H9c2 cells were lysed using Mammalian Protein Extraction Reagent (CWBIO, Beijing, China) and removed the cells debris through centrifuging for 15 min (15,000 rpm, 4 °C). Total protein was harvested and quantified using BCA assay kit (DINGGUO, Beijing, China). For each sample, approximately 20 μg proteins was separated by SDS-PAGE and transferred to a PVDF membrane. Then PVDF membrane was blocked in 5% non-fat dry milk for 2 h, followed by incubation with primary antibodies against Caspase-3 (1:1000 dilution, #14220, Cell Signaling Technology), Bcl-2 (1:1000 dilution, #3498, Cell Signaling Technology), Bax (1:5000 dilution, ab32503, Abcam), FAIM3 (1:1000 dilution, A6320, Abclonal, China), β-actin (1:1000 dilution; #4970, Cell Signaling Technology) at 4 °C overnight. Subsequently, the membrane was incubated with a secondary antibody (1:5000) at RT for 1 h. After washes, the immunoreactivity was detected using the standard chemiluminescence (Thermo-Fisher). The bands were quantified by measuring the band intensity for each group.
3
Western Blotting Protein Analysis Protocol
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Protein samples from cells and animal tissues were lysed in Cell Lysis Buffer (Beyotime, Jiangsu, China) containing 1 mM of phenylmethanesulfonyl fluoride (PMSF, Beyotime). The concentration of the protein homogenates was determined using the BCA assay Kit (Dingguo, Beijing, China). Equal volumes of protein samples were separated by SDS-poly acrylamide gel electrophoresis and electro-transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocked with 5% non-fat milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20; pH 7.6), for 2 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4 °C. Thereafter, membranes were incubated with the secondary antibodies coupled to horseradish peroxidase (HRP) for 2 h at room temperature. Protein bands were visualized with enhanced Chemiluminescence substrate Kits (ECL, New Cell & Molecular Biotech Co, Ltd, China).
4
Cell Lysis and Protein Extraction
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The cells were harvested and lysed on ice for 15 min in lysis buffer containing 150 mM NaCl, 0.1% SDS, 1% NaMoO4, 1% NP40, 50 mM Tris-HCl (pH 7.5), and 0.02% NaN3 supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF). Protein concentration was detected by using the BCA assay kit (Ding Guo Biotechnology, Shanghai, China). Subsequently, the proteins were separated on polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were separately incubated with various antibodies.
5
Protein Expression Analysis in Stem Cells
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Appropriate cells were lysed in RIPA lysis buffer containing a cocktail of protease inhibitors (Roche) and PMSF. Total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Ding Guo Biotechnology, Cat: BCA02). Antibodies against the following proteins were used for immunoblotting: NANOG (CST, Cat: 4893), ALDH (BD Pharmingen, Cat: 611195), OCT4 (CST, Cat: 2750), CD133 (CST, Cat: 64326), SOX2 (CST, Cat: 2748), phospho-AKT (Thr308) (CST, Cat: 2965), AKT (CST, Cat: 4691), phospho-GSK-3β (Ser9) (CST, Cat: 9323), GSK-3β (CST, Cat: 9315), β-catenin (CST, Cat: 8480, and β-actin (Santa Cruz Biotechnology, Cat: sc-47778). The immunoblots were scanned using an Odyssey infrared imaging system (LI-COR). Immunolabeling was detected using the ECL reagent (Sigma). Protein expression was normalized against β-actin.
6
Co-Immunoprecipitation of TRIM59 and AnxA2
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The total proteins that had been extracted from the sample cells with RIPA lysis buffer (NanJing SunShine Biotech Co., Ltd.) were quantified using a BCA Assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) in accordance with the standard protocol. For the Co-IP, 500 µg of protein was cultivated with 1–2 µg of the antibodies specific to TRIM59 (ab178847; 1:40; Abcam) and AnxA2 (ab189473; 1:30; Abcam) overnight at 4 ℃. Next, 40 µL of protein A/G PLUS-Agarose beads (Invitrogen) was added, and the proteins were cultivated at room temperature for another 2 h. Next, the beads were rinsed with lysis buffer and centrifugated at 12,000 ×g for 2 min at 4 ℃. The re-suspension of the precipitated proteins was implemented in 2× SDS-PAGE loading buffer. Finally, the immuno-complexes were analyzed by western blot.
7
Immunoblotting Analysis of Key Signaling Pathways
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Appropriate cells were lyzed in RIPA lysis buffer containing a cocktail of protease inhibitors (Roche) and PMSF. Total protein concentration was determined using the bicinchoninic acid (BCA) assay kit (Ding Guo Biotechnology, Shanghai, China). Antibodies against the following proteins were used for immunoblotting: phospho-Akt (Thr308) (#2965S , dilutions 1:1000), phospho-Akt (Ser473) (#4060S, dilutions 1:1000), Akt (#4691L, dilutions 1:1000), phospho-PDK1 (Ser241) (#3438s, dilutions 1:1000), and PDK1 (#3062, dilutions 1:1000), phospho-GSK-3β (Ser9) (#9323P, dilutions 1:1000), GSK-3β (#9315L, dilutions 1:1000), β-catenin (#8480, dilutions 1:1000), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#4370S, dilutions 1:1000), p44/42 MAPK (Erk1/2) (#4695S, dilutions 1:1000), cleaved caspase 3 (#9315L, dilutions 1:1000), all from Cell Signaling Technology (MA, USA). PARP1 (#13371-1-AP, dilutions 1:1000), PPP1R15A (#10449-1-AP, dilutions 1:500), (ProteinTech, Rosemont, IL, USA); PPP1α (#271762, dilation 1:1000) and β-actin (#47778, dilutions 1:2000), (Santa Cruz Biotechnology, Santa Cruz, CA, USA); YY1 (#61779, dilutions 1:500, Active Motif, CA, USA). The immunoblots were scanned using an Odyssey infrared imaging system (LI-COR). Immunolabeling was detected using the ECL reagent (Sigma). Protein expression was normalized against β-actin.
8
Cell Lysis and Protein Extraction
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Cells were washed twice with PBS and lysed on ice for 20 min in RIPA lysis buffer supplemented with protease inhibitors [1 mM PMSF, 1 mg/L aprotinin, 1 mg/L leupeptin, and 1 mg/L pepstatin] and phosphatase inhibitors [1 mM Na3VO4 and 10 mM NaF]. The protein concentration was measured using a BCA assay kit (Cat# BCA02, Dingguo, Beijing, China). Subsequently, the PAGE-separated proteins were transferred to PVDF membranes that were then separately incubated with indicated antibodies. The blots were visualized using a LAS 4000 instrument (GE Healthcare).
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