First, radioimmunoprecipitation assay lysis buffer was utilized for cell lysis, and after 15 min of centrifugation at 12,000 revolutions/min, the total protein concentration was assessed with bicinchoninic acid/protein detection kit (Thermo Scientific, New York, United States); 25 μg proteins were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Afterward, proteins were transferred to polyvinylidene fluoride (Bio-Rad Laboratories, Inc., United States) membrane, which was sealed with 5% skimmed milk for 1 h at room temperature and cultured overnight with primary antibodies at 4°C. Antibodies were all bought from Abcam. Primary antibodies were all rabbit antibodies: anti-CREB1 antibody, anti-PSEN1 antibody, anti–β-actin antibody, anti–E-cadherin antibody, anti–N-cadherin antibody, and antivimentin antibody. Secondary antibody was goat anti-rabbit immunoglobulin G (IgG) H&L (HRP).
Bicinchoninic acid protein detection kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bicinchoninic Acid (BCA) Protein Detection Kit is a colorimetric assay used to quantify total protein concentration in a sample. The kit uses bicinchoninic acid as the detection reagent, which changes color in proportion to the amount of protein present in the sample. This allows for the determination of protein concentration using a spectrophotometer.
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Lab products found in correlation
Cobas c702, by Roche (3 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Thermo Fisher Scientific (1 mentions)
Peroxidase labeled secondary antibody, by Abcam (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Enzyme linked immunosorbent assay kit, by Cusabio (1 mentions)
Imagequant las 4000, by Fujifilm (1 mentions)
Chemiluminescent substrate, by PerkinElmer (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Radioimmunoprecipitation lysis buffer, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Cell Signaling Technology (1 mentions)
Ab114610, by Abcam (1 mentions)
Bio spectrum gel imaging system, by Analytik Jena (1 mentions)
Ab155800, by Abcam (1 mentions)
Ab8277, by Abcam (1 mentions)
Enzyme linked immunosorbent assay kit, by R&D Systems (1 mentions)
7 protocols using bicinchoninic acid protein detection kit
1
Protein Expression Analysis by Western Blot
2
Rtn3 and GAPDH Protein Analysis
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The lung tissue was lysed by radio-immunoprecipitation assay buffer (Thermo Fisher Scientific) supplemented with protease inhibitors, after which protein level was evaluated by a bicinchoninic acid protein detection kit (Thermo Fisher Scientific). The protein was analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the protein sample on the polyvinylidene fluoride membrane was blocked, combined with primary antibodies Rtn3 and GAPDH (both from Santa Cruz Biotechnology, CA, USA), and with peroxidase-labeled secondary antibody (Abcam, CA, USA). The protein bands were analyzed with Odyssey 3.0 [23 (link)].
3
Comprehensive Metabolic and Proteinuria Analysis
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Serum creatinine (Scr), blood urea nitrogen (BUN), albumin (ALB), uric acid (UA), total protein (TP), total cholesterol (TC), triglyceride (TG) and low-density lipoprotein (LDL) were measured by Cobas C702 automatic analyzers (Roche, Basel, Switzerland). Proteinuria was measured using a bicinchoninic acid protein detection kit (Thermo Fisher Scientific, Waltham, MA, United States).
4
Biomarker Assessment of Kidney Function
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SCR and blood urea nitrogen (BUN) were measured by Cobas C702 automatic analyzers (Roche, Basel, Switzerland). Proteinuria was measured using a bicinchoninic acid protein detection kit (Thermo Fisher Scientific, Waltham, MA, United States). Serum level of diamine oxidase (DAO) were measured in accordance with the enzyme-linked immunosorbent assay kit instructions (Cusabio, Wuhan, China).
5
Protein Expression Analysis via Western Blot
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Radio immunoprecipitation lysis buffer (Thermo Fisher Scientific, Ma, USA) was applied to extract the total protein of the cells after transfection. The total protein concentration was determined using a bicinchoninic acid protein detection kit (Thermo Fisher Scientific). Proteins were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Bio‐Rad Laboratories). After blocking the membranes with the primary antibody (FLAG, Cell signaling Technology USA Cat. No. 8146; dilution of 1:1000) at 4°C overnight, they were then incubated with secondary antibody (anti‐mouse IgG, HRP‐linked antibody, CST USA Cat. No. 7076; dilution of 1:5000) at 27°C for 2 h. The target bands were finally detected using a chemiluminescent substrate (Perkin Elmer). Tanon‐5200Multi (Tanon) was used for exposure imaging. Protein expression was analyzed using the ImageQuant (LAS‐4000, FujiFilm) software. Bands were quantified using ImageJ software.
6
Western Blot Analysis of Protein Samples
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The cells were lysed with lysis buffer, and the total protein concentration was determined using a bicinchoninic acid protein detection kit (23227, Thermo Fisher Scientific). The protein samples were then diluted with 5 × sample buffer. For detection of protein in the serum samples, serum protein concentration was measured and diluted to 5 μg/uL. Then, the appropriate volume of loading buffer was added, and the mixture was boiled at 100°C for 5 min. Albumin/IgG was removed using the Albumin/IgG Depletion Kit (37591; Qiagen, Germany) according to the manufacturer's instructions. Proteins were separated for 90 min in a 12% separation gel and incubated for 1 h in a PBS blocking solution containing 5% (w/v) evaporated skimmed milk. Next, the cells were incubated with the primary antibodies against DDX17 (1 : 300, PA5-84585, Invitrogen), PDIA4 (1 : 1000, AB155800, Abcam, Cambridge, UK), and β-actin (1 : 5000, AB8277, Abcam) at 4°C, overnight. The membrane was then rinsed and incubated for 1 h with the secondary antibody (1 : 5000, ab114610, Abcam). Finally, proteins were detected using an enhanced chemiluminescence method, and images were captured using a Bio-Spectrum Gel Imaging System (UVP, Upland, CA, USA).
7
Biomarkers of Kidney Function
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Serum creatinine and blood urea nitrogen were measured by Cobas C702 automatic analyzers (Roche, Basel, Switzerland). Proteinuria was measured using a bicinchoninic acid protein detection kit (Thermo Fisher Scientific, Waltham, MA, USA). Serum levels of interleukin (IL)-6 and IL-1β were measured in accordance with the enzyme-linked immunosorbent assay kit instructions (R&D Systems, Minneapolis, MN, USA).
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