Cells were resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail, and phosphatase inhibitor cocktail, pH 7.4). Cell lysates were incubated on ice for 30 min, and cell debris was pelleted by centrifugation at 16,000 g for 30 min at 4°C. Clear lysates and cell supernatants were separated by 4–16% Bis-Tris gels (NativePAGE Novex; Thermo Fisher Scientific) and then transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked with 5% milk protein before probing with primary antibodies. Primary antibodies included anti–IL-1β (AF-401-SP; R&D Systems), anti–caspase-1 (AG-20B-0044-C100; Adipogen), and anti–β-actin (sc-1615; HRP conjugate; Santa Cruz Biotechnology, Inc.). Appropriate HRP-conjugated secondary antibodies were used, and proteins were detected using Enhanced Chemiluminescent reagent (Thermo Fisher Scientific).
Anti il 1β af 401 sp
Manufactured by R&D Systems
Sourced in United States
Anti–IL-1β (AF-401-SP) is a lab equipment product. It is a recombinant human Interleukin-1 beta (IL-1β) antibody.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Anti β actin sc 1615 hrp conjugate, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Lysis buffer, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Anti β actin sc 47778, by Santa Cruz Biotechnology (1 mentions)
2 protocols using anti il 1β af 401 sp
1
Detecting Inflammatory Biomarkers in Cell Lysates
2
Western Blot Analysis of Immune Mediators
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Mouse lung samples and BMDMs were homogenized with lysis buffer (Abcam) supplemented with protease and phosphatase inhibitors (78442, ThermoFisher), and the proteins were quantified. Samples were separated by SDS-PAGE and transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked with skim milk for 2 h, and then probed overnight with anti-GBP5 (gtx31537, Gentex, Zeeland, MI, USA), anti-iNOS (NB300-605, Novus International, St. Louis, MO, USA), anti-IL-1β (AF-401-SP, R&D Systems, Minneapolis, MN, USA), or anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBST, the blots were incubated with the appropriate anti-mouse, anti-goat, or anti-rabbit secondary antibody and then detected with ECL solution (GenDEPOT, Barker, TX, USA). Semi-quantitative densitometry was performed using ImageJ 2.1.4.6 software (National Institutes of Health, Bethesda, MD, USA).
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