Purified Mouse Anti-IKKγ Clone 54/IKKγ/NEMO (RUO)

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	-Mouse (1:2000)

Protocol tips
-Mouse (1:2000)

Publication protocol

Cells were lysed in lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 % Triton X-100, 2 mM EDTA, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM sodium orthovanadate) supplemented with protease inhibitor cocktail (Thermo Scientific), and the debris were removed by centrifugation at 10,000 × g and 4 °C. Protein concentration was determined with a micro BCA kit (Thermo Scientific). For immunoprecipitation, the samples were precleared with protein-G-Sepharose beads (Roche) for 30 minutes before immunoprecipitation with 2.5 μg antibodies and additional protein-G-Sepharose beads at 4 °C for 2 hours. Samples were then boiled in SDS sample buffer (Novex, San Diego, CA, USA) containing 10 % β-mercaptoethanol (Sigma Aldrich) and resolved by SDS-polyacrylamide gel electrophoresis. The cells were lysed and their ubiquitin conjugate content was analyzed at room temperature in denaturing conditions (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl pH 8, 1 % Triton X-100, 1 % NP-40, 20 mM imidazole). Immunoblot analysis was performed with specific antibodies and the antigen–antibody complexes were visualized by chemiluminescence (Immobilon Western, Merck Millipore, Billerica, MA, USA).

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Manufacturer protocol

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