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).
Nitrocellulose membranes
Manufactured by BD
Sourced in United States
Nitrocellulose membranes are porous sheets made from nitrocellulose material. They are used as a support medium in various laboratory techniques, such as Western blotting, dot blotting, and Northern blotting, to immobilize and transfer biomolecules, such as proteins and nucleic acids, for further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Blocking buffer, by BD (2 mentions)
Chemiluminescence detection system, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti psd95, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mitochondria isolation kit, by Thermo Fisher Scientific (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti p drp1 s616, by Cell Signaling Technology (1 mentions)
Anti cox 4, by Cell Signaling Technology (1 mentions)
P tau, by Cell Signaling Technology (1 mentions)
Anti cdk5, by Cell Signaling Technology (1 mentions)
Anti p gsk 3β, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti parp, by Cell Signaling Technology (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti drp1, by Abcam (1 mentions)
5 protocols using nitrocellulose membranes
1
Western Blot Analysis of Neurodegeneration
2
Hippocampal Protein Expression Analysis
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Protein samples of cynomolgus monkey hippocampi were harvested using the PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Korea). Equal amounts (15 μg) of protein were separated by electrophoresis on 10~15% SDS-PAGE gels and transferred onto nitrocellulose membranes (BD Biosciences, Franklin Lakes, NJ, USA). The membranes were blocked by incubation with blocking buffer (BD Biosciences) and probed with the following antibodies overnight at 4℃: anti-β-actin, anti-GFAP (Sigma-Aldrich, St. Louis, MO, USA), anti-NeuN, anti-synaptophysin, anti-PSD95, anti-phospho(p)-tau(S262), anti-p-tau(T181), and anti-p-tau(S396) (Abcam, MA, USA). Next, the membranes were washed with TBS saline containing 0.1% Tween-20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling, MA, USA) for 1 h at room temperature. After washing with TBST, the specific binding was detected using a chemiluminescence detection system (Thermo Scientific, MA, USA).
3
Western Blot Analysis of TNF-α in Hs578T Cells
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At the indicated times, Hs578T cells were harvested in ice-cold PBS and lysed on ice in a cold preparation of modified radioimmunoprecipitation buffer supplemented with protease inhibitors. Protein concentration was determined using the BCA Protein Assay Kit (Vigorous Biotechonology, Beijing, China), and equal amounts of proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide). Gels were electroblotted onto nitrocellulose membranes (BD Pharmingen). For immunoblot experiments, membranes were blocked for 2 h with 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 20, and incubated at 4°C overnight with primary antibody. Detection was performed by peroxidase-conjugated secondary antibodies using the enhanced chemiluminescence system. Primary antibodies used were GAPDH (Zhong-shan JinQiao, Beijing, China) and TNF-α (Cell Signaling Technology, Danvers, MA, USA).
4
Western Blot Analysis of Hypoxia Signaling
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Cell lysates were extracted by adding M-PER Mammalian Protein Extraction Reagent (Thermo Scientific) to the cell cultures and incubating them on ice for 20 min. After calculating the protein concentrations using a BCA protein assay kit (Thermo Scientific), the protein samples (50 µg) were subjected to 12% SDS-PAGE (Bio-Rad Laboratories) and transferred to nitrocellulose membranes (Bio-Rad, Melville, NY, USA). nitrocellulose membranes were blocked in PBS containing 0.05% Tween 20 and 3% nonfat dry milk and then incubated with a mouse monoclonal anti-HIF-1α antibody (BD Biosciences) diluted 1: 500, anti-Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) diluted 1: 1000, anti-VEGF (Abcam) diluted 1: 1000, anti-β-actin antibodies (Santa Cruz Biotechnology) overnight at 4 °C. Following three 15-min washes in TBS-T (Tris-buffered saline, 0.1% Tween 20) with 0.05% Tween 20, blots were incubated with horseradish–peroxidase-conjugated rabbit anti-mouse antibody at a 1:10,000 dilution for 2 h. The membranes were developed using enhanced chemiluminescence reagents (Thermo Scientific), and the density of bands was analyzed by Quantity One 4.6.9 software (Bio-Rad Laboratories). The protein expression levels were normalized to that of β-actin (Santa Cruz Biotechnology, Inc.) levels.
5
Protein Expression Analysis Protocol
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Total protein was prepared from homogenized tissues and cultured cells using RIPA buffer (Solarbio) plus protease inhibitors (Roche, Mannheim, Germany) and quantified by the BCA method (Thermo Fisher Scientific, Monza, Italy). Immunoblots were carried out with the isolated protein as described [14 (link)]. Equal amounts of protein were resolved by electrophoresis on Criterion™ TGX™ precast 10% gels (Bio-Rad), transferred to nitrocellulose membranes (BD Biosciences), and probed with antibodies against B cell lymphoma-2 (Bcl-2, ab182858, dilution 1:2000), vascular endothelial growth factor A (VEGFA, ab1316, dilution 1:1000), fibroblast growth factor 2 (FGF2, ab92337, dilution 1:3000), Bcl-2 associated X (Bax, ab182733, dilution 1:2000), TMED5 (ab254795, dilution 1:1000), and β-actin (ab8227, dilution 1:3000) from Abcam (Cambridge, UK). The horseradish peroxidase-conjugated IgG (ab205718, dilution 1:5000, Abcam) was used as the secondary antibody. Chemiluminescence was achieved by the incubation of Clarity ECL substrate (Bio-Rad). Protein blots were scanned and quantified using AIDA software (Raytek, Sheffield, UK).
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