The harvested cells were lysed with RIPA lysis buffer and protease inhibitor cocktail. Protein concentrations were quantified using a BCA protein assay kit. The proteins (30 µg per lane) were separated using 10% SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. The membrane was blocked with Tris-buffered saline-Tween 20 (TBS-T) and 5% nonfat dried milk for 2 h, and incubated with SIRT1 (1:500; cat. no. 19A7AB4) primary antibody overnight at 4°C. Following washing twice with TBS-T and incubation with peroxidase-conjugated secondary antibodies (1:1,000; A996702 Amyjet Scientific, Co., Ltd., Wuhan, China) for 1 h at room temperature, the bands were detected using an chemiluminescence detection system (Invitrogen; Thermo Fisher Scientific, Inc.). The band intensities were quantified using the Photo-Image System (Siemens AG, Erfurt, Germany).
Chemiluminescence detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Chemiluminescence detection system is a laboratory instrument designed to measure and analyze chemiluminescent signals. It utilizes light-emitting chemical reactions to detect and quantify a wide range of analytes, including proteins, nucleic acids, and small molecules. The system provides sensitive and accurate detection capabilities for various applications in life science research and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Pim complete, by Roche (5 mentions)
Roti quant, by Carl Roth (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti β actin, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Pvdf membrane, by Thermo Fisher Scientific (3 mentions)
Sds page, by Thermo Fisher Scientific (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Protease and phosphatase inhibitor, by Roche (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Imagequant 5, by GE Healthcare (3 mentions)
Quantity one, by Bio-Rad (2 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
Nitrocellulose membranes, by BD (2 mentions)
Blocking buffer, by BD (2 mentions)
Anti psd95, by Abcam (2 mentions)
58 protocols using chemiluminescence detection system
1
SIRT1 Protein Expression Analysis
2
Western Blot Analysis of HDACI-Treated Cells
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After treatment with HDACIs, the cells were lysed with lysis buffer (Cell Signaling, Danvers, MA) including a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed as previously reported [40 (link)]. Antibodies used for immunodetection were anti-p21 (1:1,000, Abcam, Cambridge, MA), anti-p27 (1:500, Abcam), anti-CDK2 (1:500, Thermo Scientific, Rockford, IL), anti-CDK4 (1:200, Abcam), anti-acetyl H3 (K9 + K14 + K18 + K23 + K27, 1:1,000, Abcam), anti-acetyl H3 (K27,1:2,000, Abcam), anti-acetyl H4 (K5 + K8 + K12 + K16,1:2,000, Abcam) and anti-β-actin (1:10,000, Sigma-Aldrich).
After the blot was washed, it was incubated with a horseradish peroxidase-conjugated species-specific secondary antibody (1:5,000, Jackson Laboratory, West Grove, PA) for 1 h at room temperature. The blots were developed using a chemiluminescence detection system (Invitrogen, Carlsbad, CA). Band density was analyzed using NIH ImageJ software. Densitometric measurements were performed on individual immunoblot for each antibody tested, and the values indicate the protein level normalized to the corresponding β-actin levels.
After the blot was washed, it was incubated with a horseradish peroxidase-conjugated species-specific secondary antibody (1:5,000, Jackson Laboratory, West Grove, PA) for 1 h at room temperature. The blots were developed using a chemiluminescence detection system (Invitrogen, Carlsbad, CA). Band density was analyzed using NIH ImageJ software. Densitometric measurements were performed on individual immunoblot for each antibody tested, and the values indicate the protein level normalized to the corresponding β-actin levels.
3
Renal Failure Protein Analysis
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Renal tissues were isolated from mice with chronic renal failure induced by type II diabetes and homogenized in 1X radioimmunoprecipitation assay buffer on day 60. Subsequently, western blotting was performed to analyze the expression of analyte proteins. Protein concentrations were examined using a BCA protein assay, and protein samples (40 µg) were loaded and separated using 15% SDS-PAGE. Protein were subsequently blotted on a nitrocellulose membrane and hybridized using primary antibodies against AMPK (1:2,000; 9839), phosphorylated AMPK (1:2,000; 4186), and β-actin (1:500; 3700; Cell Signaling Technologies, Inc., Danvers, MA, USA) were added for 12 h at 4°C after blocking with 5% skimmed milk for 1 h at 37°C, and membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG mAb (1:5,000; PV-6001; ZSGB-BIO, Beijing, China) for 24 h at 4°C. The blots were visualized by using a chemiluminescence detection system (32209; Invitrogen; Thermo Fisher Scientific, Inc.). Densitometric quantification of the immunoblot data was performed by using the software of Quantity-One (version 3.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Cell Lysis and Protein Analysis
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Cells were harvested in ice cold PBS and lysed in RIPA buffer (Pierce) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, total protein in the cell extracts was quantified using Rotiquant (Carl Roth). Forty micrograms of protein was resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-caspase 3, anti-beta actin (Cell Signaling), or anti COX4 (Santa cruz) primary antibodies followed by secondary horse-radish peroxidase-coupled antibodies (Rockland Immunochemicals), and signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.
5
Western Blot Analysis of p53 Protein
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Following the indicated incubation, cells were harvested in ice-cold PBS and lysed in RIPA buffer (ThermoFisher, Waltham, MA, USA) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche, Basal, Switzerland) for 20 min on ice. After centrifugation at 15,000× g for 15 min at 4 °C, the total protein in the whole-cell extracts was quantified using Rotiquant (Carl Roth, Karlsruhe, Germany). Twenty-five micrograms of protein were resolved by SDS-PAGE and blotted on polyvinylidene fluoride membranes (both Invitrogen, Carlsbad, CA, USA). The membranes were probed with primary anti-p53 and anti-β-actin antibodies, followed by anti-rabbit antibodies coupled to horseradish peroxidase (all Santa Cruz, Dallas, TX, USA). Signals were acquired in a chemiluminescence detection system (Applied Biosystems, Foster City, CA, USA) in a linear dynamic range mode.
6
Immunoblotting of OCT6 Protein
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Cells were harvested in ice-cold phosphate-buffered saline (PBS). Harvested cells were lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). In all, 40 µg of protein was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Invitrogen) and blotted on polyvinylidene fluoride membranes (Invitrogen). The membranes were probed with anti-OCT6 or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase-coupled antibodies (Rockland Immunochemicals), and signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.
7
Oxidative Stress Protein Detection
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Cells were harvested in ice-cold PBS and lysed in RIPA buffer (Cell signaling) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche) for 20 min on ice. Fifty millimolar of N-Ethylmaleimide (NEM; Sigma) was supplemented for s-glutathionylation preparations. After centrifugation at 15,000×g for 15 min at 4 °C, total protein in whole-cell extracts was quantified using Rotiquant (Carl Roth). Forty micrograms of protein were resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-GSTP1, anti-xCT, anti-catalase, anti-SOD1, anti-GPX1, anti-γGCS, or anti-β actin (Santa Cruz) primary antibodies followed by secondary horse-radish peroxidase (HRP) coupled antibodies (Santa Cruz). Signals were acquired in a chemiluminescence detection system (Applied Biosystems) in a linear dynamic range.
8
Quantifying Membrane Transporter Proteins
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Cells were harvested in ice-cold PBS and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with complete protease and phosphatase inhibitors (PIM complete; Roche, Mannheim, Germany) for 20 min on ice. After centrifugation at 15,000 × g for 15 min at 4 °C, the cell extracts’ total protein content was quantified using Rotiquant (Carl Roth, Karlsruhe, Germany). Forty micrograms of protein were resolved by SDS-PAGE (Invitrogen) and blotted on PVDF membranes (Invitrogen). The membranes were probed with anti-ASCT2, anti-β actin, and anti-LAT1 primary antibodies (Cell Signaling Technology, Danvers, Massachusetts, USA) followed by incubation with secondary horseradish peroxidase-coupled antibodies (Santa Cruz Biotechnology, Dallas, Texas, USA). Signals were acquired using a chemiluminescence detection system (Applied Biosystems, Foster City, California, USA) in the linear dynamic range.
9
Western Blot Analysis of Neurodegeneration
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Whole protein lysates were prepared using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea), and mitochondrial and cytoplasmic fractions were performed with a mitochondria isolation kit (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by electrophoresis on 8–12% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, San Jose, NJ, USA). The membranes were blocked by incubation in blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4°C: anti-NeuN, anti-AT8, anti-Tau-5, anti-β-actin (Sigma–Adrich, St. Louis, MO, USA), anti-PSD95, anti-phospho(p)-Tau(T181), anti-p-Tau(S396; Abcam, MA, USA), anti-Drp1, anti-p-Drp1(S616), anti-COXIV, anti-GAPDH, anti-PARP, anti-cleaved caspase-3, p-Tau(S262), anti-CDK5, anti-ERK, anti-p-ERK, anti-GSK3β, anti-p-GSK3β(S9; Cell Signaling, MA, USA), and anti-p35 (Thermo Fisher Scientific, Waltham, MA, USA). The membranes were washed with TBS with 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling) for 1 h at room temperature. After washing with TBST, specific binding was detected using a chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA).
10
Western Blot Analysis of Apoptosis Markers
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Western blot was used to detect the expression levels of Cleaved caspase-3, pro-caspase-3 and Bcl-2. Cells were harvested after intervention and cell pellets were lyzed with 2×sample buffer (130 mM Tris/HCl, 6% SDS, 10% 3-mercapto-1,2-propanediol, 10% glycerol). Proteins (40 μg) were subjected to 12% of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After separation, the target proteins were transferred to a nitrocellulose membrane. After blocking, the membranes were incubated overnight at 4°C with diluted antibodies against Cleaved caspase-3 (dilution 1:1000), pro-caspase-3 (dilution 1:1000) and Bcl-2 (dilution 1:1000). Blots were washed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:3000). Proteins were visualized using a chemiluminescence detection system (Thermo Scientific, USA).
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