For enrichment of ubiquitinated proteins, 4 × 106 cells were stimulated with LPS (100 ng mL−1) or R848 (1 μg mL−1), washed three times in cold PBS containing 25 mM β-Glycerophosphate disodium, 10 mM NaF and 1 mM NaVO4, and lysed for 10 min on ice in 0.6 mL of TUBE lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, 10 mM iodoacetamide, Halt Protease inhibitor (Thermo-Fisher) and Halt Phosphatase inhibitor (Thermo-Fisher)). A small fraction of the supernatants was kept for analysis of the protein input, whilst the remainder were used for protein pull down. For this, 550 μL of protein lysate was added to 30 μL of TUBE2-Agarose (Life Sensors) pre-equilibrated in TUBE lysis buffer, and the mix was then incubated with gentle agitation for 3 h at 4°C. After three washes with TBS-T, the proteins were eluted with 50 μL of sample buffer (Pierce Lane Reducing Sample Buffer (Thermo-Fisher)). Immunoblots of samples eluted from the beads and their protein inputs were then carried out as described under “Immunoblot”.
Pierce lane reducing sample buffer
Manufactured by Thermo Fisher Scientific
Pierce Lane Reducing Sample Buffer is a laboratory reagent used to prepare protein samples for electrophoresis. It contains a reducing agent that helps denature and unfold proteins, allowing for more accurate separation and analysis.
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4 protocols using pierce lane reducing sample buffer
1
Enrichment of Ubiquitinated Proteins
2
MYD88 Co-Immunoprecipitation Protocol
3
MYD88 Co-Immunoprecipitation Protocol
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MYD88 co-immunoprecipitations were performed as described previously (Tan and Kagan, 2018 (link)). Briefly, 4 × 106 cells were stimulated with LPS (100 ng mL−1) or R848 (1 μg mL−1), washed three times in cold PBS containing 25 mM β-Glycerophosphate disodium, 10 mM NaF and 1 mM NaVO4, and lysed for 10 min on ice with 0.6 mL of IP lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 2 mM dithiothreitol (DTT), 1% NP-40, Halt Protease inhibitor (Thermo-Fisher) and Halt Phosphatase inhibitor (Thermo-Fisher)). The lysates were then centrifuged at 14,000 × G for 10 min at 4°C, and the supernatants collected. A small fraction of the supernatants was kept for analysis of the protein input, whilst the remainder (550 μL) were used for co-immunoprecipitation. 1 μL of anti-MyD88 (R&D Systems, AF3109) and 30 μL of Protein G Sepharose 4 Fast Flow (Millipore Sigma) pre-equilibrated with IP lysis buffer was added to each sample. The mix was then incubated with gentle agitation for 4 h at 4°C, washed three times with IP lysis buffer, and eluted with 50 μL of sample buffer (Pierce Lane Reducing Sample Buffer (Thermo-Fisher)). Immunoblots of eluted samples and protein inputs were then carried out as described under “Immunoblot”.
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Enrichment of Ubiquitinated Proteins
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