Liver tissue (100 mg) was homogenized in 1 ml RIPA buffer (Thermo Fisher Scientific, Inc.) containing 10 µl phenylmethylsulfonyl fluoride, and centrifuged at 6,700 × g at 4°C for 5 min. The middle protein layer was taken to measure the protein concentration using a bicinchoninic acid kit (Bio-Rad Laboratories, Inc.). The samples were diluted to 50 µg/µl, mixed with sample buffer (4:1) and heated at 100°C for 5 min. SDS-PAGE (12%) was carried out and 10 µg of protein was loaded per lane and proteins were transferred onto a PVDF membrane. Membranes were blocked in TBS-0.05% Tween (TBST) containing 5% skim milk for 1 h at 25°C. After blocking, the membrane was incubated at 25°C for 2 h with primary antibodies against Cu/Zn-SOD (cat. no. PA5-270240; 1:1,000), Mn-SOD (cat. no. LF-MA0030; 1:1,000) and beta-actin (cat. no. MA5-15739; 1:1,000). The membrane was then washed with TBST and incubated at 25°C for 1 h with secondary antibody (cat. no. A32723; 1:500) (17 (link)). All antibodies were supplied by Thermo Fisher Scientific, Inc. SuperSignal™ West Pico Plus was used to visualize proteins by iBright FL1000 (both Thermo Fisher Scientific, Inc.). ImageJ version 1.44 (National Institutes of Health) was used for semi-quantitative analysis of protein expression.
Bicinchoninic acid kit
Manufactured by Bio-Rad
Sourced in United States
The Bicinchoninic acid (BCA) kit is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. The kit employs the bicinchoninic acid detection method, which involves the reduction of copper(II) to copper(I) by protein in an alkaline medium. The resulting purple-colored reaction product is measured spectrophotometrically at 562 nm, allowing for the accurate quantification of protein levels.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Gapdh antibody, by ZenBio (2 mentions)
Il 9 antibody, by Abcam (2 mentions)
D lactate antibody, by Abcam (2 mentions)
Horseradish peroxidase hrp labeled anti rabbit antibody, by Beyotime (2 mentions)
Hrp labeled anti mouse antibody, by Beyotime (2 mentions)
Heat inactivated fbs, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Ibright fl1000, by Thermo Fisher Scientific (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence kit, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Hepes, by PromoCell (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
16 protocols using bicinchoninic acid kit
1
Western Blot Analysis of Antioxidant Enzymes
2
Perihematoma Tissue Protein Analysis
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The perihematoma tissues were gathered and protein was extracted by RIPA lysis buffer. Protein concentration in the samples was determined by a bicinchoninic acid kit (Bio-Rad, Hercules, CA). Equal protein (100 μg) was loaded and separated by 8% SDS-PAGE gels, and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA) at 200 mA for 90 min. After blocking by 5% non-fat milk, the membranes were incubated by the following primary antibodies at 4 °C overnight: RNF34 (1:500) (OriGene Technologies, Beijing, China), PGC-1α (1:800) (Abcam, Cambridge, MA), uncoupling protein 2 (UCP2), manganese-dependent superoxide dismutase (MnSOD) and GAPDH (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Detection was performed using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ). GAPDH was used as an internal loading control. For immunoprecipitation assay, protein lysates were pre-cleared with A/G agarose beads (Santa Cruz Biotechnology) for 2 h and then co-incubated with A/G agarose beads and RNF34 antibody at 4 °C overnight. The immunoprecipitated protein was determined by western blotting.
3
Western Blot Protein Analysis Protocol
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Cells were washed with PBS and lysed in a lysis buffer containing 50 mM HEPES (Promocell, Heidlberg, Germany), 1% Triton X-100, and protease and phosphatase inhibitors (Pierce Biotechnology, Rockford, IL, USA). Cellular protein concentration from total cell lysates (20 μg) was quantified using a bicinchoninic acid kit (Bio-Rad, Hercules, CA, USA). Samples containing equal amounts of protein were electrophoresed on 6%–8% SDS-PAGE gels and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk in TBST (in mmol/L, 10 Tris-HCl, 150 NaCl, 0.05% Tween-20, pH 7.6) and probed with the indicated primary antibodies (1:1,000) at 4°C overnight. Then the membranes were washed with TBST three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Billerica, MA, USA) for 1 h. The membranes were exposed to enhanced chemiluminescence kit according the manufacturer’s instructions (Beyotime Institute of Biotechnology). Image quantification was performed by ImageJ software (Version 1.41; NIH, Maryland, MD, USA).
4
Western Blot Protein Quantification
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HBVSMCs or cerebrovascular tissues were lysed using RIPA buffer (Beyotime) containing 1% protease and phosphatase inhibitors (Merck). Nuclear proteins were extracted using a Nuclear/Cytosol Fractionation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The protein concentration of each sample was determined using a bicinchoninic acid kit (BioRad). Equal amounts of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 8–10% gels and then transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% non-fat milk powder in TBST consisting of 10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20 (pH 7.6) and incubated with the appropriate antibodies at 4 °C overnight. Afterwards, the blots were visualized with HRP-conjugated secondary antibodies followed detection with a chemiluminescence system (Thermo Fisher Scientific). Image quantification was performed using ImageJ software.
5
Quantifying Apoptotic Protein Expression
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A549/DDP cells were lysed using radioimmunoprecipitation assay buffer (50 mM Tris, 150 mM NaCl, 1% NPNP-40, 1% sodium deoxycholate and 0.05% SDS; pH 7.4) following co-culture with OMT-treated DCs/DC-T and treatment with DDP. Total protein was extracted and subsequently quantified using a bicinchoninic acid kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). For western blotting, protein loading was set at 40 µg and an 12% SDS-PAGE gel was used for protein separation. The proteins were transferred to a nitrocellulose membrane and incubated with primary antibodies against FOXP3 (1:3,000; cat. no. ab191416), apoptosis regulator BAX, (Bax; 1:1,000; cat. no. ab32503), B cell lymphoma-2 (Bcl-2; 1:1,000; cat. no. ab32124) and β-actin (1:1,000; cat. no. ab8224) overnight at 4°C, and subsequently incubated with Goat Anti-Rabbit (1:1,000; cat. no. ab6702) or Anti-Mouse (1:1,000; cat. no. ab6708) (all from Abcam, Cambridge, UK) secondary antibodies for 1 h at room temperature. Chemiluminescence was used to develop the color of the membrane using an ECL kit according to the manufacturer's instructions (cat. no. FD8030; Guangzhou Fude Biological Technology Co., Ltd., Guangzhou, China). ImageJ 8.0 software (National Institutes of Health, Bethesda, MD, USA) was used to perform densitometric analysis.
6
Immunoblotting Analysis of Intestinal Proteins
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For immunoblotting, the intestinal tissue was lysed in radio immunoprecipitation assay lysis buffer. The lysate was separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The protein concentration of lysate was detected using a bicinchoninic acid kit (Bio-Rad Laboratories, Hercules, CA, USA). The blot of lysates was incubated with the following antibodies: IL-9 antibody (Abcam, diluted in 1:1000), D-lactate antibody (abcam, diluted in 1:1000), zonula occludens 1(ZO-1) antibody (proteintech, diluted in 1:1000), horseradish peroxidase (HRP)-labeled anti-rabbit antibody (Beyotime, Shanghai, China), GAPDH antibody (ZEN-bio, diluted in 1:5000), and HRP-labeled anti-mouse antibody (Beyotime, Shanghai, China). Images were acquired with Tanon enhanced chemiluminescence substrate (Tanon, Shanghai, China), using Tanon 5200 imaging system (Tanon, Shanghai, China).
7
Western Blot Analysis of Cellular Proteins
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As we did before,13 (link) FaDu cells were collected for protein extraction 48 hours after siRNA transfection or 72 hours after cisplatin treatment. The FaDu cell lysates were lysed with 2× sodium dodecyl sulfate (SDS) sample buffer (100 mM Tris-HCl [pH 6.8], 10 mM ethylenediaminetetraacetic acid, 4% SDS, 10% glycine). After protein quantification with Bicinchoninic Acid kit (Bio-Rad Laboratories Inc., Hercules, CA, USA), 20 µg proteins were loaded, separated on a 10% SDS-PAGE gel, and transferred to a polyvinylidene fluoride membrane. After blocking with 5% non-fat milk for 1 hour at room temperature, the membranes were incubated with anti-TCN1 (1:1,000, Abcam), anti-PARP (1:1,000), anti-cleaved-caspase 3 (1:500), and anti-GAPDH (1:300) overnight at 4°C. The membranes were then washed with TBST three times for 5 minutes each time. Then the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. After washing three times, the signals were detected using ECL kit (Bio-Rad Laboratories Inc.). Each experiment was repeated three times.
8
Aortic Protein Expression Analysis
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The aorta samples were homogenized in lysis buffer and total protein was extracted. A bicinchoninic acid kit was used to quantify proteins (Bio-Rad, Rockford, IL, USA). After dissolving 100 μg protein sample/well, proteins were separated using 9% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with skim milk (5%, dissolved in phosphate-buffered saline) for 2 h. Primary antibodies against the following antigens were added: FGF21 (1:1000; Abcam, Cambridge, UK), NLRP3 (1:1000; Abcam), caspase-1 (1:1000; CST, Boston, MA, USA), GSDMD (1:1000; Abcam), IL-1β (1:1000; Abcam), and GAPDH (1:10000; Abcam); the blots were incubated overnight at 4°C. The blots were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG as the secondary antibody (1:5000; Calbiochem, San Diego, CA, USA) for 1 h at 20°C–25°C, washed thrice with TBS-T, developed using an ECL luminescence kit, and imaged. ImageJ software was used to analyze the protein bands. GADPH was used as an internal reference.
9
Western Blot Analysis of Cell Cycle Regulators
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Cells were lysed in radio-immunoprecipitation assay cell lysis buffer (Thermo Fisher Scientific) to extract total protein. The protein concentration was examined using a bicinchoninic acid kit (Bio-Rad, Inc., Hercules, CA, USA). Then, the protein was separated on 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% non-fat milk for 2 h and then incubated with the primary antibodies against proliferating cell nuclear antigen (PCNA, ab29, Abcam), CDC20 (#PA5-63103, Invitrogen, Thermo Fisher Scientific), CDC7 (ab229187, Abcam), cyclin A1 (CCNA1, #PA5-16519, Invitrogen, Thermo Fisher Scientific), GABPB1 (ab88746), telomerase reverse transcriptase (TERT, ab32020, Abcam) and GAPDH (ab8245, Abcam) at 4°C for 2 h, and then incubated with HRP-labeled secondary antibody (ab6721, Abcam) at 25°C for 1 h. The protein blots were developed using an enhanced chemiluminescence kit (Millipore, Corp. Billerica, MA, USA), and the protein expression was quantified using the Image J software.
10
Western Blot Analysis of Cytoskeletal Proteins
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For analysis of cytoskeletal proteins, untreated and MNPs@SiO2(RITC)-treated cells were lysed in a protease inhibitor cocktail (Sigma-Aldrich) containing radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), and total protein concentrations were determined by the bicinchoninic acid kit (Bio-Rad, Hercules, CA, USA). Proteins were separated using 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then washed three times with 0.1% Tween-20 (Sigma-Aldrich) in tris-tween buffered saline (T-TBS) and blocked with 5% non-fat milk/T-TBS. The membranes were incubated with mouse monoclonal antibodies against phosphorylated Src (p-Src), total Src (tSrc), phosphorylated FAK (p-FAK), total FAK (tFAK), phosphorylated MLY2 (p-MLY2), total (tMLY2), phosphorylated paxillin (p-PXN), total PXN (tPXN), β-actin (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), and anti-rabbit FSCS1 (ab126772) (1:10,000; Abcam) as primary antibodies. After washing with T-TBS three times, the membranes were incubated with mouse Ig kappa binding protein conjugated with horseradish peroxidase (1:1000; Santa Cruz Biotechnology). Blots were developed using an enhanced chemiluminescence solution (Thermo Fisher Scientific), and luminescence was captured on medical blue X-ray films (Agfa, Mortsel, Belgium) in a dark room.
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