Cells were lysed using lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Triton-X100, 1 mM EDTA, 10 mM NaF, 5 mM Na3VO4 and protease inhibitors). Clear supernatants were incubated with indicated antibodies for 3–4 h to overnight at 4 °C followed by isolation of immunocomplexes using protein G Sepharose 4B beads (Amersham, Uppsala, Sweden). Beads were washed three times with lysis buffer before eluting the immuno-complexes with 2 × sample buffer, run through sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and subjected to immunoblotting using specific antibodies.
Protein g sepharose 4b beads
Manufactured by Cytiva
Sourced in Sweden
Protein G Sepharose 4B beads are a type of chromatography resin used for the purification of antibodies and other proteins. The beads are composed of cross-linked agarose and covalently coupled with recombinant Protein G, a bacterial cell wall protein that binds to the Fc region of immunoglobulins. These beads provide a high-affinity and selective platform for the capture and isolation of antibodies from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using protein g sepharose 4b beads
1
Immunoprecipitation and Immunoblotting Protocol
2
NLRP3 Inflammasome Activation Assay
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Primed THP-1 cells were activated by 5 mM ATP and 1 μM nigericin for 6 h. The cells were collected and lysed at 4 °C in IP buffer containing 0.5% Nonidet-P40, 0.5% Triton X-100, 50 mM HEPES, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10% glycerol, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche), followed by centrifugation. The lysates were precleared by incubation with protein G Sepharose 4B beads (Amersham Biosciences) for 2 h at 4 °C, followed by centrifugation. Anti-ASC, anti-NRLP3 and anti-actin (SantaCruz) were incubated with protein G Sepharose 4B beads for 2 h at 4 °C. Precleared lysates were incubated with antibody-coated beads for 24 h at 4 °C. The beads were recovered by centrifugation and washed 3 times with IP buffer, and the immunoprecipitated proteins were eluted in Laemmli sample-loading buffer for subsequent immunoblot analysis.
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