Plasma membranes of RAW264.7 macrophages were harvested following a previously published protocol.8 (link),16 (link) Briefly, RAW264.7 macrophages in a good growth state were washed with PBS (HyClone) three times and then collected (centrifugation at 800 × g for 5 min). The cells were then suspended in a hypotonic solution containing 30 mM Tris-HCl, 225 mM d-mannitol, 75 mM sucrose and 0.2 mM EGTA (all reagents purchased from Sigma). The cells were then disrupted with 20 passes using a dounce homogenizer. After this step, all of the cells were ruptured. Then, the suspension solution was centrifuged at 20,000 × g for 25 min at 4 °C. The precipitate was discarded, and the supernatant was transferred to a new tube and centrifuged again at 100,000 × g for 35 min at 4 °C. After centrifugation, the supernatant was discarded, and the pellet contained the macrophage membranes. The obtained macrophage membranes were stored for a short time at −80°C. The membrane protein content was quantified with a BCA assay (Beyotime). In addition, we also extracted whole protein and nuclear protein from the cells with appropriate protein extraction kits (Sangon Biotech) and quantified the protein extracts with a BCA assay (Beyotime).
Bca assay
Manufactured by Beyotime
Sourced in China, United States
The BCA assay is a colorimetric detection method used for the quantitative determination of total protein concentration in a sample. It is based on the bicinchoninic acid (BCA) reaction, which produces a purple-colored complex that absorbs light at 562 nm. The assay is widely used in various applications, including biochemistry, cell biology, and protein research, to measure protein content accurately and precisely.
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Lab products found in correlation
Pvdf membrane, by Merck Group (110 mentions)
Ripa lysis buffer, by Beyotime (76 mentions)
Ripa buffer, by Beyotime (49 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (17 mentions)
Polyvinylidene difluoride membrane, by Merck Group (15 mentions)
Polyvinylidene fluoride membrane, by Merck Group (15 mentions)
Pvdf membrane, by Roche (13 mentions)
Protease inhibitor cocktail, by Roche (12 mentions)
Pvdf membrane, by Bio-Rad (12 mentions)
Ecl detection system, by Thermo Fisher Scientific (11 mentions)
β actin, by Cell Signaling Technology (10 mentions)
Ripa buffer, by Merck Group (10 mentions)
Nitrocellulose membrane, by Merck Group (10 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Protease inhibitor cocktail, by Merck Group (9 mentions)
Gapdh, by Abcam (9 mentions)
Gapdh, by Proteintech (9 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (9 mentions)
Ab6721, by Abcam (8 mentions)
β actin, by Abcam (8 mentions)
596 protocols using bca assay
1
Isolation of Plasma Membranes from RAW264.7 Macrophages
2
Caspase-3 Colorimetric Activity Assay
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Caspase-3 activity was determined using a colorimetric Caspase-3 assay kit (Abcam). 5x106 MCFs were collected and resuspended in 50 µl of lysis buffer and incubated on ice for 15 min, subsequently 50 µl of the 2X reaction buffer was added to each sample. DEVD-pNA substrate (1 mM) was added, and samples were incubated for 2 h at 37˚C. Caspase-3 activity was measured at an optical density (OD) of 400 nm. Protein concentration was assayed by BCA assay (Beyotime Institute of Biotechnology), BCA assay and protein concentration were performed as aforementioned. Caspase-3 activity was normalized to the protein concentration.
3
Western Blotting Quantification Protocol
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The procedure for Western blotting was as follows: the total protein was first extracted from the cell using a Protease inhibitor and a phosphatase inhibitor (Beyotime Biotechnology) and the concentration of the protein was determined by a BCA assay. The total proteins were extracted from the cells using protease inhibitor and phosphatase inhibitors (Beyotime Biotechnology) and centrifuged for 20 minutes at 12,000 g/min. The supernatant was collected and the concentration of the proteins was determined by BCA assay (Beyotime Biotechnology). Then, after electrophoresis and membrane transfer, the protein on PVDF membrane was incubated with the first antibody of GYS2 (1:500, Sigma), P53 (1:1000, Cell Signaling Technology), P21(1:1000, Cell Signaling Technology), and Cyclin D1(1:1000, Cell Signaling Technology) overnight at 4°C for 1 hour at room temperature and β-Actin (1:5000, cell signaling technology) was used as reference. The membrane was washed 3 times with TBST, and then incubated with secondary antibody at room temperature for 1 hour. The obtained bands used ECL to visualize and quantify using Gel-Pro analyzer.
4
Western Blot Analysis of Proteins in HCC
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Protein from fresh HCC samples were obtained with RIPA lysis buffer added with protease inhibitors. After quanti cation with bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China), we separated each protein through 10% SDS-PAGE and then moved them onto PVDF membranes (Millipore, USA). Then, samples were blocked with 5% nonfat milk. After incubation with primary antibodies against GAPDH and JAG2 (1:200, ab109627, Abcam, UK) and secondary antibodies, protein levels were detected with Image J (GE Healthcare Life Sciences). Each sample was analyzed three times.
The total cellular protein and the nuclear protein were extracted according to instructions of nuclear and cytoplasmic extraction reagents kit (Beyotime, Jiangsu, China). The protein concentrations were determined by bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China). 20 μg of protein was loaded per lane and separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). GAPDH was used as internal control. Antibody information: PCNA (1:1000, ab92552, Abcam, UK), MMP2
(1:1000, ab92536, Abcam, UK), MMP9 (1:1000, ab76003, Abcam, UK), Vimentin (1:1000, ab8979, Abcam, UK).
The total cellular protein and the nuclear protein were extracted according to instructions of nuclear and cytoplasmic extraction reagents kit (Beyotime, Jiangsu, China). The protein concentrations were determined by bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China). 20 μg of protein was loaded per lane and separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). GAPDH was used as internal control. Antibody information: PCNA (1:1000, ab92552, Abcam, UK), MMP2
(1:1000, ab92536, Abcam, UK), MMP9 (1:1000, ab76003, Abcam, UK), Vimentin (1:1000, ab8979, Abcam, UK).
5
Protein Extraction and Western Blot Analysis
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Total protein was extracted from LX2 cells with NP-40 lysis buffer which contained a phosphatase inhibitor cocktail (1: 100) and protease inhibitor PMSF (1: 100). After quantification by the BCA assay (Biyuntian Biotechnology, Shanghai, China), the equal protein was separated using sulfate-polyacrylamide gel electrophoresis, then transferred to the polyvinylidene fluoride membrane. Then the membrane was blocked by 5% skim milk for 1 h at room temperature and incubated with the indicated primary antibody, including COL1A1, α-SMA, p65, p-p65, GAPDH, and β-tubulin, then incubated with HRP-conjugated secondary antibodies. The corresponding bands were visualized by electrogenerated chemiluminescence (ECL). Immunoblot intensities were quantified by densitometric analysis (Image J, rsb.info.-nih.gov/ij ).
6
Protein Expression Analysis in Ileal Tissue
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Ileal tissue samples were analyzed by western blot to determine the protein levels of TLR4, LYZ, ATF6 and CHOP. The samples were flushed with PBS and homogenized in RIPA lysis buffer that contained protease inhibitor. Protein concentration was determined by BCA assay (Biyuntian, China). Equal amounts of protein were loaded, and electrophoresis was applied on sodium dodecyl sulfate-polyacr-ylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes (Millipore), incubated overnight with anti-TLR4 (1:500, ThermoFisher PA5-23,124), anti-LYZ (1:1000, ThermoFisher PA5-114,441), anti-ATF6 (1:1000, Invitrogen PA5-20,215), anti-CHOP (1:1000, ThermoFisher MA5-32,571) and β-actin (1:2000, Zhongshanjinqiao TA-09) at 4 °C overnight. Then, the membranes were incubated with Horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h at room temperature. Band intensities were semi quantitatively analyzed by ImageJ software, using β-actin bands as loading controls to normalize values.
7
Protein Expression Analysis by Western Blot
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Cells were lysed in RIPA Buffer (50 Mm Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 5 mM DTT, 2% SDS), and the protein concentration was determined using a BCA assay (Beyotime Institute of Biotechnology). Total protein (30 µg) was resolved using 10% SDS-PAGE gel, electro-transferred to polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc.) and blocked with 5% non-fat dry milk at 25°C for 1 h in Tris-buffered saline (pH 7.5). Membranes were immunoblotted overnight at 4°C with rabbit monoclonal antibodies at a dilution of 1:1,000, including keratin 5 (cat. no., 25807), E-cadherin (cat. no., 3195), vimentin (cat. no., 5741) and GAPDH (cat. no., 2118), all purchased from CST Biological Reagents Co., Ltd. A HRP-conjugated anti-rabbit IgG antibody was used as the secondary antibody (dilution, 1:2,000; cat. no., 7074; CST Biological Reagents Co., Ltd.). The incubation was performed at 25°C for 1 h. Signals were detected using enhanced chemiluminescence reagents (EMD Millipore). Signal intensities were obtained using ImageJ software (v1.51, National Institutes of Health).
8
Protein Expression Analysis of Frozen Kidney Tissue
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Kidney tissue stored at −80 °C was thawed and a portion (approximately 60 mg) was placed in RIPA lysis buffer containing and PMSF inhibitor for homogenization on ice for 30 min. After centrifugation at 14,000 rpm for 5 min at 4 °C, the protein concentration of the supernatant was determined using a BCA assay (Beyotime, Shanghai, China). After adding loading buffer and denaturation at 100 °C for 5 min, approximately 50 μg of total proteins per sample were separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% dried skimmed milk, the membranes were washed with 0.2% Tween-20 PBS and incubated overnight at 4 °C with primary detection antibodies (anti-ABCG2, anti-NLRP3 and anti-GAPDH). The membranes were then incubated with a horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Bands were visualized by enhanced chemiluminescence and analyzed using the Protein Simple Imaging system. The integrated optical density of each band was analyzed using ImageJ.
9
Mitochondrial Isolation and COX Activity
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Cells transfected with recombinant plasmids were lysed and the mitochondria were isolated using the mitochondrion isolation kit for mammalian cells (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein concentrations were measured using the bicinchoninic acid (BCA) assay (Beyotime) and COX activity was determined using the Cytochrome c Oxidase Assay kit (GenMed, Shanghai, China) as previously described (8 (link),9 (link)).
10
Protein Expression Analysis Protocol
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Total protein was extracted from various tissues or sorted cells and the supernatant protein concentrations were determined using the bicinchoninic acid (BCA) assay (Beyotime, Jiangsu, China). Equal amounts of protein were separated on sodium dodecyl sulfate‐polyacrylamide (SDS‐PAGE) gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) via a wet trans blot system. The membranes were then blocked with 5% (w/v) nonfat dried milk for 1 h at 25°C, incubated with anti‐GLUT1, anti‐GLUT3, anti‐PARP, anti‐caspase 3, or anti‐β‐actin antibodies (EnoGene, Nanjing, China) at 4°C overnight, and incubated with horseradish peroxidase‐conjugated secondary antibody at room temperature for 1 h. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime, Jiangsu, China).
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