Treated cells were washed, detached with scraping, pelleted, resuspended with Nupage SDS loading buffer (ThermoFisher Sci.), and lysed with three 10s pulses at 40% sonication power (Heat Systems Ultrasonics, Plainview, NY). Lysates were loaded into NuPage 12% or 4–12% Bis-Tris gel wells (ThermoFisher Sci.) for gel electrophoresis, then transferred to nitrocellulose membrane with iBlot and developed using Inflammasome Antibody Sampler Kit #32961T (CST Danvers, MA) or rabbit anti-LTA or LT-B as previously described [46 (link)]. Blots were imaged with Pierce™ ECL Western Blotting Substrate (ThermoFisher Sci.) and Amersham Imager 600 and quantified using ImageQuant LT (GE Healthcare, Pittsburgh, PA).
Nupage sds loading buffer
Manufactured by Thermo Fisher Scientific
NuPAGE SDS loading buffer is a ready-to-use solution designed for sample preparation in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. It contains the necessary components to denature and prepare protein samples for electrophoretic separation.
Automatically generated - may contain errors
Lab products found in correlation
Nupage sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Pierce g2 fast blot, by Thermo Fisher Scientific (2 mentions)
Image lab v5, by Bio-Rad (2 mentions)
Chemidoc mp imager, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Nupage 12 or 4 12 bis tris gel wells, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Strataclean resin, by Agilent Technologies (1 mentions)
Anti caspase 1 p10 m 20, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin tub2, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
4 protocols using nupage sds loading buffer
1
Inflammasome Protein Analysis by Western Blot
2
Western Blot Protocol for Protein Analysis
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Cells were lysed using RIPA buffer, and lysates were prepared for electrophoresis with NuPAGE™ sample reducing agent (Thermofisher) and NuPAGE™ SDS loading buffer (Thermofisher). Samples were heated to 95°C for 5 minutes. A total of 25 µg of protein was resolved with SDS-PAGE using 3-8% tris-acetate gels for blots probing polyubiquitin or 4-12% bis-tris gels for all the others. Proteins were transferred to nitrocellulose membranes using Thermo Scientific™ Pierce™ G2 Fast blot. Membranes were incubated in primary antibody diluted in PBST with 2% BSA overnight at 4°C, washed, and incubated with the appropriate secondary antibody diluted in PBST with 5% skimmed milk. Blots were developed using Clarity Western ECL substrate (Bio-Rad) and imaged using ChemiDoc MP Imager (Biorad). All images were processed in ImageLab v 5.2.1 (Biorad).
3
Western Blot Analysis of Polyubiquitin
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Cells were lysed using RIPA buffer, and lysates were prepared for electrophoresis with NuPAGE™ sample reducing agent (Thermofisher) and NuPAGE™ SDS loading buffer (Thermofisher). Samples were heated to 95 °C for 5 min. A total of 25 µg of protein was resolved with SDS-PAGE using 3-8% tris-acetate gels for blots probing polyubiquitin or 4-12% bis-tris gels for all the others. Proteins were transferred to nitrocellulose membranes using Thermo Scientific™ Pierce™ G2 Fast blot. Membranes were incubated in primary antibody diluted in PBST with 2% BSA overnight at 4 °C, washed, and incubated with the appropriate secondary antibody diluted in PBST with 5% skimmed milk. Blots were developed using Clarity Western ECL substrate (Bio-Rad) and imaged using ChemiDoc MP Imager (Biorad). Images were acquired with ImageLab v 5.2.1 (Biorad) using the built-in protocol "Chemi Hi-sensitivity" via autoexposure or manual exposure. Global contrast and gamma adjustments were done on the images. All antibodies used are in Supplementary Table 3.
4
NLRP3 Inflammasome Activation Assay
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