Proteins were extracted from BMDM or ESDM in triplicate with RIPA buffer (Pierce, 89900,) as previously described (5) . 15 to 50 µg of each cell lysate was mixed with SDS sample buffer and incubated for 8 min at 95°C then immediately cooled on ice for 8 min. Proteins were separated by SDS-PAGE (ThermoFisher, XP04200BOX) for 2 hours at 110V, and then transferred to a PDVF membrane. After incubating with Casein Blocker in TBS (ThermoFisher, 37532) for 1 hour at room temperature, the membrane was incubated overnight at 4°C with primary rabbit ASC antibody (Cell Signaling, 67824) 1:500 in blocking buffer. After washing with PBS-0.05% Tween20, the membrane was probed with HRPconjugated secondary antibody (goat anti-rabbit) 1:20,000 in blocking buffer for 1 hour at room temperature. The bands were visualized by HRP chemiluminescence detection Reagent (Millipore, WBKLS0500). The membrane was re-probed with HRP-conjugated anti β-actin (Santa Cruz Biotech, sc-47778) 1:20,000 in blocking buffer for 1 hour at room temperature and visualized in the same way. Densitometric analysis of bands was performed using ImageJ software.
Hrp chemiluminescence detection reagent
Manufactured by Merck Group
Sourced in United States
The HRP Chemiluminescence Detection Reagent is a laboratory product used for the detection and quantification of horseradish peroxidase (HRP) in various applications. It utilizes a chemiluminescent reaction to generate a measurable light signal proportional to the amount of HRP present in the sample. The reagent provides a sensitive and reliable method for HRP detection without further interpretation or extrapolation.
Automatically generated - may contain errors
2 protocols using hrp chemiluminescence detection reagent
1
Western Blot Analysis of ASC Protein
2
Western Blot Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Tissues were frozen at -80°C immediately after dissection for protein extraction.
Then, they were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and0.05% SDS, pH 7.4) while broken into pieces on ice. The sample buffer was added, and the supernatant was collected after centrifugation at 12,000 rpm for 10 min at 0°C. After boiling for 10 min, the denatured proteins were separated by polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane, which was purchased from Millipore (Billerica, MA, USA). The membranes were washed with methanol for 3min prior to staining. Then, the membranes were blocked with 5% low-fat dried milk in TBS containing 0.5% Tween-20 (TBS-T) prior to a 2-h incubation at room temperature with a 1:800 dilution of an anti-human p-STAT3 (Tyr 705) antibody (Cell Signaling Technology, Beverly, MA, USA). The anti-human β-actin antibody (bzbio.com) was used to indicate the amount of proteins loaded. After fully rinsing, the membranes were incubated with an HRP-conjugated secondary antibody (bzbio.com). The membranes were developed with HRP chemiluminescence detection reagent (Millipore), and then exposed to Hyper film ECL (Thermo Scientific, Waltham, MA, USA).
Then, they were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and0.05% SDS, pH 7.4) while broken into pieces on ice. The sample buffer was added, and the supernatant was collected after centrifugation at 12,000 rpm for 10 min at 0°C. After boiling for 10 min, the denatured proteins were separated by polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane, which was purchased from Millipore (Billerica, MA, USA). The membranes were washed with methanol for 3min prior to staining. Then, the membranes were blocked with 5% low-fat dried milk in TBS containing 0.5% Tween-20 (TBS-T) prior to a 2-h incubation at room temperature with a 1:800 dilution of an anti-human p-STAT3 (Tyr 705) antibody (Cell Signaling Technology, Beverly, MA, USA). The anti-human β-actin antibody (bzbio.com) was used to indicate the amount of proteins loaded. After fully rinsing, the membranes were incubated with an HRP-conjugated secondary antibody (bzbio.com). The membranes were developed with HRP chemiluminescence detection reagent (Millipore), and then exposed to Hyper film ECL (Thermo Scientific, Waltham, MA, USA).
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