Rats' intestinal mucosa was homogenized on ice-cold water with lysis buffer (Beyotime, P0013 B). Equal protein concentrations were separated by SDS-PAGE (Beyotime, P0050) and transferred to the PVDF membrane (Beyotime, FFP24). After blocking 30 min with the QuickBlock™ Blocking Buffer (Beyotime, P0252), membranes were incubated overnight at 4°C with primary antibodies against MAPK (Abcam, ab185145), IL-1β (Abcam, ab200478), IL-6 (Abcam, ab259341), TGF-β (Cusabio, CSB-PA004279), and TNF-α (Abcam, ab66579). GAPDH (Abcam, ab181602) was used as a normalizing protein. Membranes were then washed 3 times with Western Wash Buffer (Beyotime, P0023C3) 3 times, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 2 h. Membranes were then washed 3 times with Western Wash Buffer (Beyotime, P0023C3), then incubated with luminescent substrate solution (Beyotime, P0018M). Protein bands were visualized by Chemiluminescence Imaging System (Bio-Rad, CA, USA) and analyzed with Image J software.
Ab200478
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab200478 is a primary antibody that targets a specific protein. It is designed for use in various laboratory techniques, such as immunoassays and immunohistochemistry. The product is manufactured by Abcam and is available for purchase.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ab1872, by Abcam (3 mentions)
Ab215203, by Abcam (3 mentions)
Ab181602, by Abcam (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab23914, by Abcam (2 mentions)
Ab203224, by Abcam (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Ab92726, by Abcam (2 mentions)
Bis tris protein gel, by Thermo Fisher Scientific (2 mentions)
Ab178547, by Abcam (2 mentions)
Ab82390, by Abcam (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Ab193349, by Abcam (2 mentions)
Pa1 32045, by Thermo Fisher Scientific (2 mentions)
Ab6673, by Abcam (2 mentions)
Anti alix, by Cell Signaling Technology (2 mentions)
Hpa002926, by Merck Group (2 mentions)
Anti flotillin, by Cell Signaling Technology (2 mentions)
14 protocols using ab200478
1
Western Blot Analysis of Inflammatory Markers
2
Western Blot Analysis of Sphingolipid Signaling
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Cell or tissue samples were lysed using radioimmunoprecipitation assay buffer. Protein content was quantified using a bicinchoninic acid assay kit (PAB180007, Bioswamp), and 20 μg of proteins were loaded onto a 12% gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred to polyvinylidene fluoride membranes and blocked with 5% skim milk at room temperature for 2 h. Then, the membranes were incubated overnight at 4°C with primary antibodies against S1P (rabbit, 1:1,000, ab140592, Abcam), PAR-1 (mouse, 1:1,000, NB1-71779-SS, Novus Biologicals), S1PR1 (rabbit, 1:1,000, NB120-11424, Novus Biologicals), SphK1 (rabbit, 1:1000, ab71700, Abcam), SphK2 (rabbit, 1:1,000, 1-SP030-02, Quartett), IL-1β (rabbit, 1:500, ab200478, Abcam), and GAPDH (rabbit, 1:5,000, 10494-1-AP, Proteintech, Rosemont, IL, USA). The membranes were then washed three times with PBS/Tween 20 for 3 min each and incubated at room temperature for 1 h with goat anti-rabbit IgG (1:10,000, PAB150011, Bioswamp) or goat anti-mouse IgG (1:10,000, PAB150009, Bioswamp) secondary antibodies. After three washes in PBS/Tween 20 for 5 min each, the membranes were incubated with an enhanced chemiluminescence reagent (WBKLS0010, Millipore) in the absence of light and visualized using an automatic analyzer (Tanon-5200, Tanon, Shanghai, China).
3
Protein Isolation and Quantification
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Total protein was isolated from cells by RIPA (Sigma-Aldrich) on ice. The supernatants were centrifuged at 12 000g for 10 minutes at 4°C before the collection of the supernatant proteins. The concentration of protein in each sample was measured by the BCA kit (Sigma-Aldrich). Each protein sample (15 µg) was separated by the SDS-PAGE and transferred onto a PVDF membrane. Then the PVDF membrane was blocked by 5% fat-free milk at 37°C for 1 hours, incubated with primary antibodies against IL-1β (ab200478; RRID:AB_2888939; 1:1,000; Abcam), TNF-α (ab205587; RRID:AB_2889389; 1:1,000; Abcam), MAPK6 (ab53277; RRID:AB_2140288; 1:1,000; Abcam) and GAPDH (ab181602; RRID:AB_2630358; 1:1,000; Abcam) at 4°C overnight; and HRP-conjugated goat anti-rabbit secondary antibody (ab7097; RRID:AB_955411; 1:3,000; Abcam) at 37°C for 1 hours, successively. At last, the protein bands were visualized by ECL Kit (Pierce). Immunodetection was analyzed by Image Lab version 3.0 (Bio-Rad Laboratories, Inc.). IL-1β, TNF-α, and MAPK6 were referred to as GAPDH.
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from tissues by RIPA lysis buffer (Beyotime), 10 μL PMSF and 10 μL phosphatase inhibitor. SDS-PAGE gel electrophoresis was utilized to separate the extracted protein. The protein was transferred to polyvinylidene fluoride membrane. After blocking with 50 g/L BAS for 1 h, the membrane was incubated with primary antibodies against HTR2B (1/3000; ab227722; Abcam, Cambridge, MA, United States), SLC5A3 (1/1000; ab113245), GAPDH (1/3000; ab8245), TNF-α (1/1000; ab215188), IL-1β (1/1000; ab200478), TGF-β (9ab208156) and IL-4 (1/1000; ab34277) at 4°C overnight. After washing the membrane with TBST, the membrane was incubated with secondary antibody (1/5000; ab7097) for 1.5 h. The ultra-sensitive ECL chemiluminescence kit was used to configure the luminescence buffer, and the band was developed.
5
Western Blot Analysis of Inflammation and Antioxidant Markers
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Total protein was extracted with RIPA buffer (Beyotime, China). Then the concentrations in the samples were determined with BCA (Beyotime, China) methods. Next, the protein samples were separated with 10% SDS-PAGE gel (Beyotime, China). After that, the proteins were transferred to the PVDF membrane (Millipore, United States). Then, the membranes were blocked with a 5% skim milk powder solution (BD Bioscience, Regilait, France). After that, the membranes were incubated overnight with primary antibodies at 4°C. The primary antibodies used in this study were TNF-α (ABCAm, ab34674), IL-1β (ABCAm, ab200478), IL-18 (ABCAm, ab71495), NRF-2 (ABCAm, ab137550), HO-1 (ABCAm, ab137749) and GAPDH (ABCAm, ab181602). On Day 2, the membranes were washed with PBST three times. The membranes were then incubated with the second antibody for 2 hours. Finally, bands were developed with enhanced chemiluminescence reagents (Millipore, United States).
6
Antibodies for Adipogenesis and Inflammation
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Antibodies against UCP1 (ab10983), PGC1α (ab72230), PRDM16 (ab106410), IL1β (ab200478), IL6 (ab7737), TNFα (ab8348), TLR4 (ab13556), NLRP3 (ab210491), BMP4 (ab155033), p-Smad1 (ab73211), p65NF-κB (ab16502), IĸBα (ab32518), and GAPDH (ab9484) were from Abcam (Cambridge, England). The experimental procedure was described in our previous reports in detail (26 (link)).
7
Western Blot Characterization of Extracellular Vesicles
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Exosomes or cell pellets were lysed in Cell Lysis Buffer (Cell Signaling) with protease and phosphatase inhibitor cocktail (Millipore). All lysates were cleared by centrifugation, and quantified by bicinchoninic acid (BCA) protein assay (ThermoFisher). 5 µg of protein from each sample were electrophoresed on 4–12% Bis-Tris Protein gels, and transferred to nitrocellulose membranes (ThermoFisher). Blots were probed with primary antibodies: anti-Angiopoietin-2 (2948, Cell Signaling), anti-WISP1 (ab178547, Abcam), anti-PDGF BB (ab23914, Abcam), anti-IL-1beta (ab200478, Abcam), anti-FN (1574-1, Epitomics), anti-KLF4 (HPA002926, Sigma), anti-GFP (ab6673, Abcam), anti-β-actin (8457s, Cell Signaling), anti-CD81 (Eat2 Thermo Scientific), anti-CD63 (ab193349, Abcam), anti-mouse CD9 (ab82390, Abcam), anti-human CD9 (ab92726, Abcam) , anti-Flotillin (18634, Cell Signaling), anti-Alix (2171, Cell Signaling), anti-Annexin V (8555, Cell Signaling), LAMP2 (ab203224, Abcam), anti-HSP70 (4876, Cell Signaling), anti-EpCAM (2626, Cell Signaling), and anti-GM130 (12480, Cell Signaling), then probed with the secondary antibody anti-rabbit IgG-HRP (7074s, Cell Signaling), or anti-mouse IgG-HRP (7076, Cell Signaling), or anti-Armenian hamster (PA1-32045, Thermo Scientific). HRP-based detection was performed using SuperSignal West Substrate (Thermo Scientific).
8
Western Blot Analysis of Inflammatory Markers
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Total protein was extracted from treated cells using 1% RIPA Lysis Buffer (Beyotime, Jiangsu, China) with a phosphorylation inhibitor (Roche, Indianapolis, USA). The total protein concentration for each sample was measured with a bicinchoninic acid (BCA) kit (Beyotime). Equal amounts of protein samples were then subjected to SDS-PAGE and proteins were then transferred to PVDF membranes (Beyotime). The membranes were blocked with 5% skim milk at room temperature for 1 hr and incubated with the primary antibodies against LOX-1 (1:1,000, ab60178, Abcam), NLRP3 (1:1,000, ab4207, Abcam), ASC (1:5,000, ab127537, Abcam), caspase1 (1:1,000, ab1872, Abcam), mature IL-1β (1:1,000, ab200478, Abcam) and β-actin (1:1,000, ab8226, Abcam). Subsequently, the membranes were incubated in anti-rabbit IgG horseradish peroxidase conjugated antibody (1:5,000; Abcam) for 1 hr at room temperature. The immune complexes were detected using the SuperSignal west pico kit (Shanghai solarbio Bioscience & Technology, China). The intensities of the signals were quantified using ImageJ software 6.0.
9
Western Blot Characterization of Extracellular Vesicles
10
Investigating Signaling Pathways in Neuroinflammation
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NQDI-1 was purchased from Selleck Chemical Inc. (Houston, TX). Antibodies for p-p38 (Tyr182) (1:800, #9211S), ASK1 (1:1000, #8662S), pERK1/2 (Thr202/Tyr204) (1:1000, #4370), and p-JNK (Thr183/Tyr185) (1:1000, #9255S) and CGRP antibody (1:100, #14959) were purchased from Cell Signaling Technology (Beverly, MA). Antibody for GAPDH (1:5000, G9545) was purchased from Sigma-Aldrich Inc. (St. Louis, USA). Antibody for p-ASK1 (Thr845) (1:1000, bs-3031R) was purchased from Bioss (Woburn, MA). Antibodies for IBA-1 (1:100, ab178847), GFAP (1:100, ab7260), IL-1β (1:1000, ab200478), and TNF-α (1:1000, ab6671) were purchased from Abcam (Cambridge MA). Anti-mouse IgG, HRP-linked Antibody (1:3000, #7076) and Anti-rabbit IgG, HRP-linked Antibody (1:3000, #7074) were purchased from Cell Signaling Technology (Beverly, MA). All other chemicals were purchased from Sigma Chemical Co (St. Louis, MO).
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