Western blot was performed as previously described.34 , 35 CAF derived from patients with NPC and NPC cells treated with CM were lysed in radioimmunoprecipitation assay buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktails (Roche, Basel, Switzerland) on ice, and the protein levels were quantified by using BCA protein assay kit (Dingguo Biotech Co., Beijing, China), according to the manufacturer's instruction. The total protein was separated with 10 to 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analyzed by Western blot analysis.
Bca protein assay kit
Manufactured by Dingguo
Sourced in China
The BCA protein assay kit is a colorimetric detection and quantification method for proteins. It utilizes the bicinchoninic acid (BCA) reaction to measure the total protein concentration in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Luminata crescendo western hrp substrate, by Merck Group (8 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Quantity one software, by Bio-Rad (5 mentions)
A600669, by Sangon (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ecl reagent, by Thermo Fisher Scientific (4 mentions)
Hy k0022, by MedChemExpress (3 mentions)
Odyssey system, by LI COR (3 mentions)
Fusion fx5 image analysis system, by Vilber (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Hy k0010, by MedChemExpress (2 mentions)
Protease and phosphatase inhibitor cocktail, by MedChemExpress (2 mentions)
Iseq00010, by Merck Group (2 mentions)
Ai600 imaging system, by GE Healthcare (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
63 protocols using bca protein assay kit
1
Western Blot Analysis of NPC Cells
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared in ice-cold RIPA lysis buffer (Beyotime, Haimen, China) and the protein concentrations of the samples were determined using a BCA protein assay kit (Dingguo, Beijing, China). Equal amounts of proteins were separated by SDS-PAGE electrophoresis, transblotted on PVDF membrane and probed with rabbit anti-REST polyclonal antibody (1:1000, Millipore, Temecula, CA, USA), rabbit anti-CCND1 antibody (1:1000, Abcam, Cambridge, MA, USA), rabbit anti-CCNE1 antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-BBC3 polyclonal antibody (1:1000, Affinity Biosciences, Zhenjiang, China), rabbit anti-DAXX polyclonal antibody (1:1000, Affinity Biosciences, Zhenjiang, China) and mouse anti-β-actin antibody (1:5000, Proteintech, Rosemont, IL, USA). After incubation with HRP-conjugated secondary antibodies (1:5000, KangChen, Shanghai, China), protein bands were visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Princeton, NJ, USA) on Tanon-5200 chemiluminescence detection system. The bands were analyzed by using Image J software (NIH, Bethesda, MD, USA).
3
Protein Quantification and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were quantified using the BCA Protein Assay Kit (Dingguo, China), and then the same amount of protein samples were added to SDS-PAGE to separate proteins, followed by Coomassie blue staining. The proteins were transferred to PVDF membrane (Bio-Rad, USA), blocked with 5% skim milk for 1 h and incubated with the primary antibodies and followed by the secondary antibodies for 1 h at room temperature before imaging with ECL Western Blotting Substrate (Solarbio, China).
4
Quantifying Inflammatory Cytokines in Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and interleukin-1β (IL-1β) were analyzed by their respective commercial kits (Tianjin Anorikang Biotechnology Co., Tianjin, China) using serum or supernatants of liver obtained by homogenizing with saline (1:9, w/v) followed by centrifugation at 10,000 × g for 10 min at 4 °C. The protein concentration in the tissue was determined by a BCA protein assay kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., Beijing, China), and the result was expressed as pg/mg protein.
5
Lipid Profile Quantification in Serum and Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The contents of triglyceride (TAG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) in the serum and liver were assayed by their respective commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. For liver samples, the tissue was homogenized in ice-cold anhydrous ethanol (1:9, w/v) and then centrifugated at 10,000 × g for 10 min at 4 °C. The protein concentration in the resulting supernatant was determined by a BCA protein assay kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., Beijing, China). TAG, TC, HDL-c, and LDL-c concentrations in the serum and liver were expressed as mmol/L and mmol/g protein, respectively.
6
Oxidative Stress Biomarkers in Liver and Intestine
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The contents of nitric oxide (NO), malondialdehyde (MDA), and reduced glutathione (GSH) and the activities of inducible nitric oxide synthase (iNOS), superoxide dismutase (SOD), and myeloperoxidase (MPO) in the liver and small intestine were assessed by corresponding commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) using the supernatant of the tissue obtained by homogenizing with saline (1:9, w/v) followed by centrifugation at 10,000 × g for 10 min at 4 °C. The protein concentration in the tissue was determined by a BCA protein assay kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., Beijing, China), and the result was expressed as μmol/g protein or nmol/g protein or U/mg protein. Plasma levels of iNOS, NO, and GSH were evaluated by the same method.
7
SKOV3 Cell Lysis and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SKOV3 cells were lysed in RIPA buffer (CWBIO, China) containing protease inhibitor cocktail (CWBIO, China) and phosphatase inhibitor cocktail (CWBIO, China) and incubated on ice for 20 min. Protein concentrations were determined using a BCA protein assay kit (DingGuo, China). Total protein extracted from SKOV3 cells were separated by 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, USA). After blocking in 5% non-fat dry milk in TBS at room temperature for 1 h, the membranes were incubated with primary antibodies including cleaved caspase3, caspase3, and P21 (Cell Signaling, Denver, MA) and β-actin (Proteintech, USA) overnight at 4°C. Subsequently, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibody (Proteintech, USA) for 1 h at room temperature. Finally, the protein expression was imaged using a gel image analysis system (Bio-Rad, USA).
8
Detailed Methodology for Cell Line Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chemical markers (purity ≥98%) were purchased from Sichuan Victory Biological Technology Co., Ltd. Materials for tissue culture were purchased from Invitrogen (Carlsbad, CA). The antibodies were obtained from Abcam (Cambridge, United Kingdom), Absin (Absin Bioscience Co., Ltd.) and Invitrogen. BD GentestTM ATPase assay kit was purchased from BD Bioscience (San Jose, CA). GST activity assay kit, BCA protein assay kit, A549 cell lines were purchased from Dingguo Changsheng Biotechnology Ltd. (Guangzhou, China). A549-DDP (cisplatin resistance) cell was purchased from Fudan IBS cell bank (FDCC). TRIzol™ Reagent, High-Capacity cDNA Reverse Transcription Kits, PowerUp™ SYBR™ Green Master Mix, Pierce® Fast Western Blot Kit, and PVDF Transfer Membrane were purchased from ThermoFisher Scientific (Guangzhou, China). Materials that not specified here were purchased from Sigma-Aldrich. (St. Louis, MO), ThermoFisher Scientific, Merck (Whitehouse Station, NJ), Dingguo and Xinjing Biotechnology.
9
Biochemical Markers in Metabolic Disorders
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Triacylglycerols (TAGs), total cholesterol (TC), low-density cholesterol (LDL-C), high-density cholesterol (HDL-C), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and malonaldehyde (MDA) levels were measured spectrophotometrically according to the instructions of the kit (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China). Protein concentration was determined using a BCA protein assay kit (Dingguo Changsheng Biotech, China). The 4-hydroxynonenal (4-HNE) activity was determined using a Hailian Biotechnology Co. Ltd. ELISA (enzyme-linked immunosorbent assay) kit (Jiangxi, China).
10
Protein Expression Analysis in Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular protein was extracted from liver tissues using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was measured using a BCA protein assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). To assess the protein expression levels of Bax, Bcl-2, and caspase 3 proteins, 80 µg of protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were incubated at 4°C overnight with primary antibodies against caspase 3 (1:800, 9662, Cell Signaling Technology, Inc., Danvers, MA, USA), Bax, Bcl-2 and GAPDH (1:500; sc-70407, sc-23960 and sc-32233; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and subsequently with a secondary horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse antibody (1:4,000, both Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein bands were visualized using an enhanced chemiluminescence detection kit (Thermo Fisher Scientific, Inc.) and exposed to X-ray film. Protein expression was normalized to the levels of GAPDH.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!