Selected article for: "ice lysis buffer and lysis buffer"
Author: Leznicki, Pawel; Korac-Prlic, Jelena; Kliza, Katarzyna; Husnjak, Koraljka; Nyathi, Yvonne; Dikic, Ivan; High, Stephen
Title: Binding of SGTA to Rpn13 selectively modulates protein quality control
  • Document date: 2015_9_1
    • ID: 1pi9nccc_26
    Snippet: For pull-down experiments from mammalian cell lysate, HeLa cells were transiently transfected with the indicated plasmids using GeneJuice (Merck) according to manufacturer's instruction. After 24 h cells were lysed in ice-cold lysis buffer [50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 25 mM NaF, 10 mM ZnCl2] freshly supplemented with complete protease inhibitor cocktail (Roche) and, after a pre.....
    Document: For pull-down experiments from mammalian cell lysate, HeLa cells were transiently transfected with the indicated plasmids using GeneJuice (Merck) according to manufacturer's instruction. After 24 h cells were lysed in ice-cold lysis buffer [50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 25 mM NaF, 10 mM ZnCl2] freshly supplemented with complete protease inhibitor cocktail (Roche) and, after a pre-clearing centrifugation step (20 min, 13,000×g, 4°C), the soluble fraction was incubated for 4 h at 4°C with recombinant proteins immobilised on Glutathione Sepharose resin. Beads were washed with lysis buffer, bound proteins eluted with SDS sample buffer and subjected to SDS-PAGE and western blotting.

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