To investigate the effects of genes on intracellular acetyl-CoA and triglycerides, ICP2 cells were treated with a final concentration of 160 μmol/L sodium oleate (Sigma) for 48 h after transfection. ICP2 cells were then washed twice with PBS and lysed with the lysis buffer. After ultrasonication, the cell mixture was resuspended by centrifugation at 8,000 × g for 10 min at 4 ºC. The intracellular acetyl-CoA content was determined using an Acetyl Coenzyme A Content Assay Kit (Boxbio, Beijing, China), according to the manufacturer’s instructions. The ICP2 cells were then washed twice with PBS, and lysis buffer was added for ultrasonication. Thereafter, the cell suspension was incubated for 10 min at 70 °C and centrifuged at 2,000 × g for 5 min at 25 °C. The intracellular triglyceride content was detected using a Tissue Triglyceride (TG) Content Assay Kit (Applygen, Beijing, China), according to the manufacturer’s instructions. The intracellular total protein content was assessed using a BCA Protein Quantification Kit (Applygen) to normalize the acetyl-CoA and triglyceride content.
Bca protein quantification kit
Manufactured by Applygen
Sourced in China
The BCA protein quantification kit is a laboratory equipment used for the colorimetric detection and quantification of protein concentrations. It is based on the bicinchoninic acid (BCA) method and provides a simple, reliable, and sensitive way to measure protein levels in various samples.
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Lab products found in correlation
Sodium oleate, by Merck Group (1 mentions)
Tissue triglyceride tg content assay kit, by Applygen (1 mentions)
Alphaview 3, by Bio-Techne (1 mentions)
Enhanced chemiluminescence plus system, by GE Healthcare (1 mentions)
Goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Methanol activated polyvinylidene difluoride membranes, by Merck Group (1 mentions)
Membrane and cytosol protein extraction kit, by Beyotime (1 mentions)
Gel imager, by Bio-Rad (1 mentions)
β actin, by Applygen (1 mentions)
Sodium hydrosulfide, by Merck Group (1 mentions)
L cysteine, by Sinopharm (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Arg 1, by Abcam (1 mentions)
Ripa lysis buffer, by Applygen (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using bca protein quantification kit
1
Measuring Intracellular Acetyl-CoA and Triglycerides
2
Protein Extraction and Quantification
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Total protein was extracted and its concentration was assessed by using the BCA protein quantification kit (Applygen, Beijing, China). The total protein (50 μg) was separated on a 10% SDS-PAGE gel and transferred to methanol-activated polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% bovine serum albumin blocking solution for 1 h at room temperature and incubated with primary pan-acetyl-lysine antibody (PTM-101; 1:1000 dilution), primary pan-succinyl-lysine antibody (PTM-419; 1:1000 dilution), and primary pan-malonyl-lysine antibody (PTM-902; 1:6400 dilution; Jingjie Biotechnology Co, Hangzhou, China) overnight at 4°C, respectively. Subsequently, the membranes were washed with PBS-T and incubated with peroxidase conjugated secondary antibodies goat anti-mouse IgG (1:10,000 dilution; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence plus system (GE Healthcare, Marlborough, MA, USA), and optical density of the bands were analyzed by AlphaView 3.0 (Alpha Innotech, San Jose, CA, USA).
3
Molecular Mechanisms of Cardiovascular Regulation
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BCA protein quantification kit (Applygen), sodium hydrosulfide (Sigma-aldrich), L-cysteine (Sinopharm), propargylglycine (Aladdin), Anti-eNOS Rabbit Antibody (Santa Cruz), Anti-p-Akt Mouse Antibody (Santa Cruz), Anti-t-Akt Mouse Antibody (Santa Cruz), Anti-cPKCβII Rabbit Antibody (Santa Cruz), HRP-labeled Goat Anti-Mouse IgG (Applygen), HRP-labeled Goat Anti-Rabbit IgG (Applygen), β-actin, Mouse mAb (Applygen), Multi-functional enzyme immunoassay (Thermo), Gel imager (Bio-Rad), Membrane and cytosol protein extraction kit (Beyotime).
4
Western Blot Analysis of Protein Expression in Lung Tissue
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Total protein was extracted from frozen lung tissue using RIPA Lysis Buffer (Applygen, Beijing, China) supplemented with protease/phosphatase inhibitor cocktail. The protein concentrations were determined with a BCA protein quantification kit (Applygen, Beijing, China). The lysates were mixed with 8% SDS-PAGE and then electrophoresis was performed. The proteins were then transferred onto polyvinylidene fluoride membranes (Bio-Rad) and were blocked with 5% skim milk for 2 h at room temperature. The membranes were incubated with primary antibodies against a-SMA (1:1000), Arg1 (1:1000), OTC (1:1000), CPS1 (1:2000), ASS1 (1:1000), and GAPDH (1:40,000, all from Abcam, Cambridge, MA, United States) overnight at 4°C. Subsequently, the blots were incubated for 1 h at room temperature with secondary antibodies (1:5000; Abcam). The protein bands were detected using electrochemiluminescence (ECL) reagent (Tanon, Shanghai, China) and the intensities of the signals were quantitatively analyzed using ImageJ Software.
5
Western Blotting Analysis of Protein Expression
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Western blotting analysis was performed according to the method described in our previous report [32 (link)] with a minor modification. Briefly, cells were washed twice with PBS, disrupted on ice for 30 min in NP-40 (50 nM Tris (pH 7.4), 1% NP-40, 150 mM NaCl and 40 mM NaF) or RIPA lysis buffer (Thermo Scientific) supplemented with protease and phosphatase inhibitors (Pierce Chemical) and cleared by centrifugation. Protein concentration was determined with a BCA protein quantification kit (Applygen, Beijing, China). Equal amount of protein (35 μg) in cell lysates was separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, immunoblotted with specific primary antibodies at a 1:1000 dilution, second antibodies at a 1:2000 dilution and detected by chemiluminescence with the ECL detection reagents (Amersham Biosciences).
6
Intracellular Cholesterol Quantification in Chicken Hepatocellular Carcinoma Cells
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The cell culture medium was removed, and the chicken hepatocellular carcinoma cells were washed with PBS for 3 times before incubated with 0.25% (w/v) Trypsin, 0.53 mM EDTA solution at 37°C for 2–5 min. After centrifuging cells at 1,100 rpm, the supernatant was discarded and 500 μl PBS was added. Cell breakage was accomplished and the intracellular total cholesterol (T-CHO) was determined using cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer’s instruction. An appropriate amount of fresh cellular compounds was used to measure total protein concentration to normalize T-CHO level using BCA Protein Quantification Kit (Applygen, Beijing, China). All experiments were performed in triplicate, and independently repeated three times. The standard curves for intracellular T-CHO and total protein measurement were calculated using the internal standards following the manufacturer’s instructions.
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