HEK293T were transfected using CaPO4 and 24 h later, treated with 1 μM MCC950 or 60 μM acalabrutinib for 6 h or with 60 μM ibrutinib for 2 h followed by 4-h incubation with medium without ibrutinib, where indicated. Cells were lysed 48 h after transfection in RIPA buffer supplemented with protease/phosphatase inhibitors (Roche). Lysates were subjected to immunoprecipitation of the NLRP3-HA or NACHT-FLAG fusion protein with Dynabeads (Sigma-Aldrich) or with agarose beads covered with PI4P (P-B004a; Echelon Biosciences). Washed beads were boiled in loading buffer and applied to standard SDS-PAGE on Thermo Fisher Scientific precast gels, followed by immunoblot according to the antibody manufacturer’s instructions. Membranes were exposed using Fusion FL camera and FusionCapt Advance software (PEQLAB). Quantification was conducted using the same software.
Fusioncapt advance software
Manufactured by Avantor
FusionCapt Advance software is a data acquisition and analysis tool designed for laboratory equipment. It provides real-time monitoring and control of various instruments and devices. The software enables users to collect, visualize, and analyze data from connected equipment.
Automatically generated - may contain errors
Lab products found in correlation
Fusion fl camera, by Avantor (2 mentions)
Fusion fx instrument, by Avantor (2 mentions)
Dynabeads, by Merck Group (1 mentions)
Protein g dynabead, by Thermo Fisher Scientific (1 mentions)
λ phosphatase p0753, by New England Biolabs (1 mentions)
Protease phosphatase inhibitor, by Roche (1 mentions)
Plin2, by Proteintech (1 mentions)
Hrp coupled secondary antibody, by Abcam (1 mentions)
Nupage 4 12 bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
0.45 m nitrocellulose membrane, by GE Healthcare (1 mentions)
Odyssey clx instrument, by LI COR (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
4 protocols using fusioncapt advance software
1
NLRP3 Inflammasome Regulation by Kinase Inhibitors
2
Immunoprecipitation and Immunoblotting of Phosphorylated Proteins
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PBMCs or BMDMs were primed with LPS and stimulated with nigericin, washed with cold PBS, and immediately lysed in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease/phosphatase inhibitors (Roche). A sample of the cleared lysate was taken before addition of the primary antibody (see Table S2 ). To selected samples, 2,000 U λ-phosphatase (P0753; New England Biolabs) and MnCl2 to 1 mM were added and incubated for 30 min at 30°C. After 18 h of rotation at 4°C, magnetic bead–coupled secondary antibody (Protein G Dynabeads; Thermo Fisher Scientific) was added for another 90 min. The beads were then washed three times with lysis buffer, resuspended in SDS loading buffer, and boiled. Standard SDS-PAGE was performed on Thermo Fisher Scientific precast gels followed by immunoblot according to the antibody manufacturer’s instructions. Membranes were exposed using Fusion FL camera and FusionCapt Advance software (PEQLAB). Quantification was conducted using the same software.
3
PLIN2 Protein Expression Analysis
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Cells were lysed in 1 × RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% IGEPAL (NP-40), 0.1% SDS, 1 mM EDTA, and 0.5% Na-Deoxycholate) with protease inhibitors. Twenty microgram of protein was analyzed by western blot with antibodies against PLIN2 (Proteintech) and ACTIN (CST). HRP coupled secondary antibodies were obtained from Abcam. Chemiluminescence was detected on a Fusion FX instrument (PeqLab) and analyzed with Fusion Capt Advance software (PeqLab) using rolling ball background correction.
4
Western Blot Analysis of Protein Expression
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Frozen cell pellets were lysed in 1x RIPA buffer (Sigma-Aldrich) with protease and phosphatase inhibitors (Roche, Sigma-Aldrich). 20 µg of protein were loaded into nupage 4–12% bis-tris precast gels (Thermo Fisher Scientific) and run with MES buffer. Proteins were transferred to a 0.45 µm nitrocellulose membrane (GE healthcare). Membranes were blocked with 5% milk in TBS/0.1% Tween (TBST) for 1 h at RT. Antibodies were diluted as described in Table S2 . Incubation with primary antibodies was performed overnight at 4°C. Membranes were washed three times with TBST and secondary antibody incubation was performed for 1–2 h at RT followed by washing as above. In case of HRP coupled secondary antibodies, chemiluminescence was detected on a Fusion FX instrument (PeqLab). For detection of β-actin an IR dye 680 coupled secondary antibody (LICOR) was used and detection was performed on an Odyssey CLx instrument (LI-COR). Analysis was performed with Fusion Capt Advance software (PeqLab) using rolling ball background correction or with Image Studio light 5.2 software (LI-COR).
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