Total protein was isolated from MPC5 cells using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and quantitated using a BCA protein quantification kit (Abcam, Cambridge, UK), and the proteins were separated via 10% SDS‒PAGE. Then, the proteins were transferred to PVDF membranes (Roche, Basel, Switzerland). After being sealed with 5% nonfat milk and washed three times with TBST, the membranes were incubated with primary antibodies against Bax (1:1,500, ab182733; Abcam, Cambridge, UK), Bcl-2 (1:2,000, ab182858; Abcam, Cambridge, UK), α-SMA (1:400, MA5-14084; Thermo Fisher Scientific, Waltham, MA, USA), podocin (1:800, PA5-79757; Thermo Fisher Scientific, Waltham, MA, USA), synaptopodin (1:1,000, ab259976; Abcam, Cambridge, UK), p-p65 (1:800, MA5-15160), NLRP3 (1:800, ab263899), cleaved caspase-1 (1:1,000, PA5-99390; Thermo Fisher Scientific, Waltham, MA, USA), GSDMD (1:900, ab209845; Abcam, Cambridge, UK), and β-actin (1:8,000, MA1-140; Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. After being washed three times with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:7,000) for 1 h, and then the immunoblots were visualized using an ECL kit (Roche, Basel, Switzerland) and quantitated using ImageJ software (Bethesda, Rockville, MD, USA).
Ab182733
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Ab182733 is a recombinant protein that serves as a positive control for Western blotting applications. The product is a recombinant form of the target protein, which can be used to validate the specificity of primary antibodies and ensure the accuracy of Western blot results.
Automatically generated - may contain errors
Lab products found in correlation
Ab182858, by Abcam (27 mentions)
Ab32124, by Abcam (20 mentions)
Ab9485, by Abcam (18 mentions)
Pvdf membrane, by Merck Group (14 mentions)
Ab205718, by Abcam (12 mentions)
Ripa lysis buffer, by Beyotime (11 mentions)
Ab2302, by Abcam (10 mentions)
Ab196495, by Abcam (9 mentions)
Ab8227, by Abcam (9 mentions)
Ab8245, by Abcam (9 mentions)
Ripa buffer, by Beyotime (9 mentions)
Ab32042, by Abcam (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Ab214430, by Abcam (7 mentions)
Bca kit, by Beyotime (7 mentions)
Ab59348, by Abcam (7 mentions)
Ab6721, by Abcam (6 mentions)
Ab194583, by Abcam (6 mentions)
Ab184787, by Abcam (6 mentions)
Ab181602, by Abcam (5 mentions)
91 protocols using ab182733
1
Quantitative Analysis of Apoptosis and Inflammation Markers
2
Protein Expression Analysis in Cells
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The total protein was obtained by cells lysis with RIPA Lysis Buffer (Haigene, Harbin, China), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose (NC) membrane. Then, the membrane was placed in 5% milk for 2 h at room temperature, washed in phosphate buffer saline (PBS). The B-cell lymphoma-2 (Bcl-2, ab32124), Bcl-2 associated X protein (Bax, ab182733), cleaved caspase-3 (ab2302), β-actin (ab179467) and THBS2 (ab84469) antibodies from Abcam were diluted 1,000 times and added into membranes to incubate at 4°C overnight. And then, anti-rabbit IgG antibody (1:2,000; Abcam) was added to incubate for 30 min at room temperature. Finally, the proteins on the membrane were detected by an imaging system (Bio-Rad, Hercules, USA).
3
Extraction and Characterization of MO-B from Ophiopogon japonicus
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Ophiopogon japonicus was obtained from farms in Cixi (Zhejiang, China). MO-B was extracted from Ophiopogon japonicus using high-speed counter-current chromatography (19 (link)) and the yield was ~0.2–0.4 mg/g in tuber roots of Ophiopogon japonicus. High-performance liquid chromatography (HPLC) was conducted to measure the purity of MO-B, which was >97% (Fig. S1 ).HPLC was performed using a Shimadzu C18 column (5 µm 250×4.6 mm). The volume ratio of mobile solvents A (water) and B (acetonitrile) was maintained at 35:65, and the temperature was set at 30°C. The flow rate of the mobile phase was 1 ml/min. The detection wavelength was 285 nm. MO-B was then dissolved to 10, 20, 40 and 50 µM in dimethyl sulfoxide for cell treatment.
Antibodies targeting Bax (ab182733), Bcl-2 (ab182858), cleaved caspase-3 (ab32042), neutrophil cytochrome b light chain (p22phox; ab80896) and GAPDH (ab9482), goat anti-mouse horseradish peroxidase IgG (ab6789) and goat anti-rabbit IgG horseradish peroxidase (ab6721) secondary antibodies, were purchased from Abcam. Cell Counting Kit-8 (CCK-8), ROS and malondialdehyde (MDA) detection kits, radioimmunoprecipitation assay (RIPA) lysis buffer, a BCA Protein Assay kit and superoxide dismutase (SOD) assay kit with WST-8 were purchased from Beyotime Institute of Biotechnology.
Antibodies targeting Bax (ab182733), Bcl-2 (ab182858), cleaved caspase-3 (ab32042), neutrophil cytochrome b light chain (p22phox; ab80896) and GAPDH (ab9482), goat anti-mouse horseradish peroxidase IgG (ab6789) and goat anti-rabbit IgG horseradish peroxidase (ab6721) secondary antibodies, were purchased from Abcam. Cell Counting Kit-8 (CCK-8), ROS and malondialdehyde (MDA) detection kits, radioimmunoprecipitation assay (RIPA) lysis buffer, a BCA Protein Assay kit and superoxide dismutase (SOD) assay kit with WST-8 were purchased from Beyotime Institute of Biotechnology.
4
Engeletin Neuroprotection in Stroke
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We resuspended engeletin (>99.0% pure, MW = 434.39) in dimethyl sulfoxide (DMSO). For dose–response studies, rats were randomly assigned to six different treatment groups: sham group, tMCAO group, edaravone group (6 mg/kg) and engeletin groups (15, 30 and 60 mg/kg). Antibodies (HMGB1: ab79823.html">ab79823 , TLR4: ab22048.html">ab22048 , Bcl‐2: ab32124, NF‐κB p65: ab16502, Bax: ab182733 and Cleaved Caspase‐3: ab214430) were acquired via Abcam (Cambridge, MA, United States), and anti‐GAPDH (AF2819) from Beyotime Biotechnology (Shanghai, China).
5
Western Blot Analysis of Autophagy and Apoptosis
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Western blot assay was performed following the standard protocol. In brief, after total protein was isolated and quantified, the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel (SDS-PAGE; Solarbio, Beijing, China) and then transferred onto polyvinylidene difluoride membranes (PVDF; Pall Corporation, New York, NYC, USA). Next, the membranes were blocked in slim milk for 2 h and then incubated with primary antibodies against ATG3 (ab108251; 1:2000; Abcam, Cambridge, MA, USA), LC3Ⅱ/LC3Ⅰ (ab128025; 1:200; Abcam), P62 (ab56416; 1:500; Abcam), B-cell lymphoma-2 (BCL-2; ab196495; 1:1000; Abcam), cleaved caspase-3 (ab49822; 1:500; Abcam), cleaved caspase-9 (ab2324; 1:2000; Abcam), BCL2-Associated X (Bax; ab182733; 1:2000; Abcam) or GAPDH (ab181602; 1:5000; Abcam) at 4°C overnight followed by incubation with HRP-conjugated secondary antibody (D110150; 1:5000; Sangon, Shanghai, China) for 2 h. The protein bands were analyzed using an enhanced chemiluminescence reagent (Beyotime).
6
Western Blot Analysis of Apoptosis Regulators
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The protein extraction from brain tissues or bEnd.3 cells was quantified by BCA protein assay kit (Beyotime Biotechnology, Shanghai, China), denatured at 98 °C for 10 min, and then, separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies against B-cell lymphoma-2 (Bcl-2) (ab194583, 1 : 1000 dilution, Abcam, Cambridge, UK), Bcl-2-associated x protein (Bax) (ab182733, 1 : 2000 dilution, Abcam), FOXO3 (ab70315, 1 : 5000 dilution, Abcam) and β-actin (ab8227, 1 : 5000 dilution, Abcam). Subsequently, the membranes were incubated with secondary antibody (ab6721, 1 : 10 000 dilution, Abcam), conjugated by horseradish peroxidase for 2 h at room temperature and interacted with enhanced chemiluminescence chromogenic substrate (Beyotime Biotechnology). The protein signals were analyzed with Image Lab software (Bio-Rad) with β-actin as the loading control.
7
Western Blotting Analysis of Apoptosis Regulators
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After 48 h of transfection, the cells were lysed on ice and the total protein was extracted. After measuring the concentration of proteins, protein samples (30 μg) were separated by 10% SDS-PAGE and subsequently transferred onto PVDF membranes. The membrane was blocked with TBST containing 5% skim milk for 1 h. Later, membrane was incubated with primary antibodies including CEP55 rabbit polyclonal antibody (ab170414, 1:5000, abcam, Cambridge, UK), Bax rabbit polyclonal antibody (ab182733, 1:1000, abcam, Cambridge, UK), Bcl-2 rabbit polyclonal antibody (ab32124, 1:1000, abcam, Cambridge, UK) and GAPDH rabbit polyclonal antibody (ab9485, 1:2500, Abcam, Cambridge, UK) overnight at 4°C. TBST was used to wash the membranes for three times with 15 min for each time. Next, the membrane was incubated with secondary antibody of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (ab6721, 1:3000, abcam, Cambridge, UK) at room temperature for 1 h. After washing the membranes with TBST, the enhanced chemiluminescence (ECL) kit (P0018FS, Beyotime) was added and ChemiDoc™ Touch Imaging System (12003153, BIO-RAD) was used for exposed photography. The experiment was repeated three times.
8
Verification of ALR IgG Purity and Apoptosis Markers
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To verify the purity of the ALR IgG, 20 µg ALR was processed for SDS-PAGE, and the bands were visualized by silver staining using the Fast Silver Stain Kit (Beyotime). For western blotting, proteins were extracted from cells using RIPA lysis buffer (Beyotime), and protein concentrations were determined with a bicinchoninic acid (BCA) Protein Assay (Beyotime). For western blotting, proteins (30 µg/well) were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes, blocked with 5% nonfat milk, and probed with ALR (1:1,000), Bax,1:2,000 (ab182733; Abcam, Cambridge, UK), Bcl-2, 1:2,000 (ab194583; Abcam), Cleaved caspase-3 (1:1,000; Cell Signaling Technology), and cleaved caspase-9 (1:1,000; Cell Signaling Technology) primary antibodies.
9
Western Blot Analysis of Ischemic Myocardium
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The extracted protein lysates from ischemic myocardial tissues and cardiomyocytes were used for Western blot analysis as described previously [27 (link)]. The lysates were normalized to equal amounts of protein, and 10 μg protein from cell lysates or 30 μg protein from tissue lysates were separated by 10% or 12% SDS–PAGE, transferred to polyvinylidene difluoride membrane, and then sealed with 5% nonfat milk in TBST for 3 h. Membranes were incubated with the following primary antibodies: p-AMPK (1:2000, #ab133448, Abcam), AMPK (1:1000, #ab80039, Abcam), PGC-1α (1:1000, #ab191838, Abcam), SIRT3 (1:1000, #ab264041, Abcam), SOD2 (1:5000, #ab13533, Abcam), Bax (1:2000, #ab182733, Abcam), B-cell-lymphoma protein 2 (Bcl-2) (1:2000, #ab182858, Abcam), caspase3 (1:2000, #ab184787, Abcam), cleaved-caspase3 (1:5000, #ab214430, Abcam), and β-tubulin (1:20,000, #66240-1-lg, Proteintech) at 4 °C overnight. Proteins were then probed with specific HRP-conjugated secondary antibodies for 30 min at room temperature. Finally, the protein bands were obtained using the ChemiDoc Touch Imaging System (Chemi Doc, Bio-Rad, Hercules, CA, USA), and the relative intensity of the bands was quantified using Image Lab software.
10
Protein Expression Analysis in HUVECs
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After extracting the proteins from HUVECs with RIPA (Thermo Fisher Scientific), they were first boiled at 98°C for 5 min to denature. The samples were separated and then transferred to polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, U.S.A.) membranes. The membranes were blocked in 5% non-fat dry milk for 2 h, and incubated with primary antibody of B-cell lymphoma-2 (Bcl-2, 1:1000, ab32124, Abcam, Cambridge, MA, U.S.A.), Bcl-2-associated X (Bax, 1:2000, ab182733, Abcam), cleaved-caspase3 (1:500, ab32042, Abcam), tumor necrosis factor-α (TNFα, 1:1000, ab6671, Abcam), IL-6 (1:1000, 12912S, Cell Signaling Technology, Shanghai, China), IL-8 (1:1000, ab110727, Abcam), IL-1β (1:1000, 12703S, Cell Signaling Technology), TRPM7 (1:1000, ab109438, Abcam) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3000, ab8245, Abcam) at 4°C overnight. The membranes were washed and incubated with secondary antibodies for 1 h, and detected by an odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, U.S.A.).
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