Brains were lysed in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% (v/v) Triton-X, 0.27 M Sucrose, HALT Protease inhibitor (ThermoFisher), 1 mM DTT, 1 mM PMSF, 100 mM NEM. Halo-tagged Ubqln1, Fam63, or Eps15 UBDs were immobilized on Halo-Link Resin (Promega) overnight at 4°C, washed. then incubated with brain lysates for 2 hr at 4°C, to immunoprecipitate proteins modified with specific ubiquitin chains. Resin was washed three times in lysis buffer, boiled in 1× NuPAGE LDS Sample Buffer and Sample Reducing Agent (Thermo Fischer Scientific), and analyzed by western blot.
Nupage lds sample buffer and sample reducing agent
Manufactured by Thermo Fisher Scientific
The NuPAGE LDS Sample Buffer and Sample Reducing Agent are laboratory products designed for sample preparation in protein electrophoresis. The LDS Sample Buffer is a lithium dodecyl sulfate-based buffer that denatures and solubilizes proteins, while the Sample Reducing Agent contains reducing agents to break disulfide bonds. These products are intended to be used as part of the sample preparation process for protein analysis using electrophoresis techniques.
Automatically generated - may contain errors
2 protocols using nupage lds sample buffer and sample reducing agent
1
Ubiquitin Chain Interactome Profiling
2
PI3 Kinase p85 Immunoprecipitation
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Cell lysates were quantified and equal amounts were subject to immunoprecipitation overnight with PI3 Kinase p85 antibody (CST) or IgG mouse (CST) isotype control and Dynabeads Protein G (Invitrogen). Immuno-complexes where washed thrice with AZ cell lysis panel buffer and resuspended in pure water and NuPAGE LDS sample buffer and sample reducing agent (Thermo Fisher Scientific).
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