Tissues were harvested and resuspended in RIPA lysis buffer (20 mM Tris-HCl pH 8.0, 60 mM NaCl, 0.2% glycerol, 0.02% NP-40, 0.04 mM EDTA) supplemented with protease inhibitors. The suspension was then sonicated for 30 seconds and subsequently centrifuged at 12,000 rpm for 15 min at 4°C. The protein concentration was measured using a BCA Protein Assay Kit (Solarbio). The total protein samples were separated on SDS-PAGE Gel and transferred to a PVDF membrane (Cytiva). Membranes were blocked for 2 hours at room temperature with blocking buffer and incubated overnight with specific primary antibodies at 4°C. The primary antibodies were anti-pro-IL-1β (Abclonal, A11370), anti-IL-6 (Abclonal, A0286), and anti-β-Tubulin (Abmart, M20005H). Bound primary antibodies were detected by HRP-conjugated secondary antibodies, donkey anti-Rabbit IgG (Cytiva, NA934V) and sheep anti-Mouse IgG (Cytiva, NA931V). Images were captured using an automated chemiluminescence imaging analysis system (Tanon).
Anti il 6
Manufactured by ABclonal
Sourced in China
Anti-IL-6 is a laboratory product that binds to and neutralizes the cytokine interleukin-6 (IL-6). It can be used in research applications to study the role of IL-6 in biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti tnf α, by ABclonal (3 mentions)
Anti il 1β, by ABclonal (2 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Anti β tubulin, by Abmart (1 mentions)
Donkey anti rabbit igg, by Cytiva (1 mentions)
Anti vegf, by ABclonal (1 mentions)
Western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Anti acsl3, by ABclonal (1 mentions)
Anti scd1, by Abcam (1 mentions)
Anti pparα, by ABclonal (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti parkin, by Proteintech (1 mentions)
Anti magl, by Abcam (1 mentions)
Anti inos, by Proteintech (1 mentions)
Anti arg 1, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
5 protocols using anti il 6
1
Western Blot Analysis of Inflammatory Proteins
2
Immunohistochemical Analysis of Membrane Tissue
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Standard IHC staining was performed on 4-μm-thick paraffin sections of the membrane tissue for the expression of BMP-2, vascular endothelial growth factor (VEGF), von Willebrand factor (vWF), transforming growth factor (TGF)-β, and interleukin (IL)-6. After deparaffinization, heat-induced epitope retrieval was performed (95°C/30 min) in citrate buffer (pH = 6.0). Commercial antibodies were adapted for IHC analysis; these included anti-BMP-2 (polyclonal, 1:200; cat. no. A0231; ABclonal, MA, USA); anti-VEGF (polyclonal, 1:200; cat. no. A0280; ABclonal); anti-vWF (vWF Picoband antibody, 1:200; cat. no. PB9062; Boster Biological Technology, Pleasanton CA, USA); anti-TGF-β (polyclonal, 1:200; cat. no. A2561; ABclonal); and anti-IL-6 (polyclonal, 1:200; cat. no. A0286; ABclonal). Endogenous peroxidase was quenched in a 0.3% hydrogen peroxide and 50% methanol solution for 10 min, followed by rinsing in phosphate-buffered saline (PBS). TAHC-04D (BioTnA, Taiwan) was employed as the signal detecting system.
3
Maternal Placental and Liver Protein Profiling
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The protein levels in maternal placenta and livers were determined by western blot analysis using anti-IL6, anti-TNF-α, anti-IL-1β, anti-PPARα, and anti-ACSL3 (all from ABclonal, Wuhan, China); anti-SCD1 (Abcam, Cambridge, MA); anti-SREBP1c (CST, Danvers, MA); and anti-FASN (CST, Danvers, MA) antibodies. Total proteins were extracted and subsequently separated by 8–12% SDS-PAGE gels. Proteins were transferred onto PVDF membranes and probed with corresponding antibodies overnight at 4°C, followed by incubation with anti-mouse or anti-rabbit secondary antibodies (Beyotime, Shanghai, China) for 1 h at room temperature. The proteins were visualized by using a Western chemiluminescent HRP substrate (Millipore Corporation, United States).
4
Western Blot Analysis of Synovial Proteins
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Western blotting was performed as previously described (28 (link)). The protein concentrations of the synovial tissues or cells were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). Proteins (30 µg) were separated by SDS-PAGE on a 10% gel and transferred to PVDF membranes (MilliporeSigma). The PVDF membranes were blocked with 5% non-fat dry milk for 2 h, followed by incubation with one of the following primary antibodies at 4°C overnight: Anti-MAGL (1:1,000, Abcam, cat. no. ab246902), anti-iNOS (1:1,000, ProteinTech Group, Inc., cat. no. 22226-1), anti-Arg1 (1:5,000, ProteinTech Group, Inc., cat. no. 16001-1), anti-TNF-α (1:1,000, ABclonal, cat. no. A20851), anti-IL-1β (1:1,000, ABclonal, cat. no. A16288), anti-IL-6 (1:1,000, ABclonal, cat. no. A11115), anti-PTEN-induced kinase 1 (PINK1) (1:1,000, ProteinTech Group, Inc., cat. no. 23274-1), anti-Parkin (1:1,000, ProteinTech Group, Inc., cat. no. 14060-1), or anti-β-actin (1:5,000, ProteinTech Group, Inc., cat. no. 81115-1). The following day, the membranes were washed and incubated with the secondary antibody (1:5,000, Abcam, cat. no. ab205718) for 2 h at room temperature. The proteins were detected using Pierce ECL Western Blotting Substrate (Thermo Fisher).
5
Protein Expression Analysis of Aortic and SMC Samples
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Human and mouse aortas or cultured SMCs were harvested and lysed using RIPA Lysis Buffer (Beyotime Biotechnology). Equal amount (15‐30 μg) of total protein was fractionated on 5%‐15% SDS‐polyacrylamide gel and transferred onto a PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA). Then the blots were incubated with primary antibodies: anti‐GDF11 (1:1000, abcam, Cambridge, MA, USA), anti‐ACTA2 (1:1000, Novus Biologicals), anti‐MMP‐2 (1:500, Proteintech, Rosemont, IL, USA), anti‐MMP‐9 (1:1000, Proteintech), anti‐MMP‐3, anti‐TNF‐α (1:500, ABclonal, Wuhan, China), anti‐IL‐6, anti‐p‐Smad‐2, and anti‐p‐Smad‐3 (1:1000, ABclonal) at 4°C overnight. The membranes were probed with species‐relevant HRP‐linked secondary antibodies (1:10 000, Proteintech) at 37°C for 40 minutes. Besides, housekeeping protein β‐actin (1:2000, Proteintech) was used as the internal control.
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