Cells were washed with PBS and lysed using RIPA buffer containing protease inhibitors. Protein concentration was measured by a detergent resistant Bradford assay, and equal amounts of protein were boiled with SDS-containing Laemmli sample buffer. Western blotting was performed following standard procedure. Primary antibodies used were anti-caspase-1 (Genentech), anti-NLRP3 (Adipogen), anti-NLRC4 (LSBio), anti-ASC (SCBT), anti-IL1b (R&D), anti-IRF8 (Biolegend), anti-IRF4 (Biolegend), anti-histone H3 (CST), or anti-β-actin (SCBT). HRP (Jackson Lab) conjugated secondary antibodies were used. Proteins were visualized by using Chemi-luminescent SuperSignal (Thermo Fisher) and ECL signal was recorded on X-ray films using a developer (Kodak).
Chemi luminescent supersignal
Manufactured by Thermo Fisher Scientific
SuperSignal is a chemi-luminescent detection reagent used for the sensitive detection of proteins in Western blot analysis. It generates a luminescent signal upon reaction with the enzymes conjugated to the detection antibodies, enabling the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti irf4, by BioLegend (2 mentions)
Anti β actin, by SCBT (2 mentions)
Anti caspase 1, by Genentech (2 mentions)
Anti nlrc4, by LSBio (2 mentions)
Anti asc, by SCBT (2 mentions)
Anti il1b, by R&D Systems (2 mentions)
Anti irf8, by BioLegend (2 mentions)
Anti nlrp3, by Adipogen (2 mentions)
Anti sirt1, by Abcam (1 mentions)
Anti h4k16ac, by Merck Group (1 mentions)
Anti phospho h2ax ser139, by Merck Group (1 mentions)
3 protocols using chemi luminescent supersignal
1
Western Blot Analysis of Inflammasome Proteins
2
Western Blot Analysis of Inflammasome Proteins
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Cells were washed with PBS and lysed using RIPA buffer containing protease inhibitors. Protein concentration was measured by a detergent resistant Bradford assay, and equal amounts of protein were boiled with SDS-containing Laemmli sample buffer. Western blotting was performed following standard procedure. Primary antibodies used were anti-caspase-1 (Genentech), anti-NLRP3 (Adipogen), anti-NLRC4 (LSBio), anti-ASC (SCBT), anti-IL1b (R&D), anti-IRF8 (Biolegend), anti-IRF4 (Biolegend), anti-histone H3 (CST), or anti-β-actin (SCBT). HRP (Jackson Lab) conjugated secondary antibodies were used. Proteins were visualized by using Chemi-luminescent SuperSignal (Thermo Fisher) and ECL signal was recorded on X-ray films using a developer (Kodak).
3
Protein Expression Analysis by Western Blot
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Protein extracts, prepared with RIPA buffer (50 mM Tris HCl (pH 7,4); 50 mM NaCl; 1% NP40 10%; 0.5% sodium deoxycolate 10%; 0.1% SDS 1%) containing protease inhibitors (PMSF 1 mM, Na3VO4 1 mM, pepstatin 10 mg/ml, leupeptin 10 mg/ml, aprotinin 10 mg/ml), were separated by electrophoresis in SDS-PAGE polyacrylamide gel and transferred to a PVDF membrane. Then, each membrane was blocked for 40 minutes in a solution with 5% of non-fat milk. Next, primary antibody was added to the membranes and incubated according to manufacturer’s instructions. The membranes were washed and the secondary antibody was incubated for one hour, according to manufacturer’s instructions. Primary antibodies used were: anti-phospho-H2AX (Ser139) (Merck Millipore, 07–164), anti-SIRT1 (Abcam, #Ab12193), anti-DNMT1 (Imgenex, IMG-261A), anti-DNMT3B (Active Motif, #39207), anti-H4K16ac (Millipore, Temecula, CA, #07–329). After secondary antibodies incubation (KPL), the membranes were washed with TBS-T, detected with chemiluminescent SuperSignal (ThermoScientific) and visualized by luminescence reading device UVITEC (Cambridge, www.uvitec.co.uk ).
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