Selected article for: "anti mouse and ph Tris HCl"
Author: Blank, Maximilian F.; Chen, Sifan; Poetz, Fabian; Schnölzer, Martina; Voit, Renate; Grummt, Ingrid
Title: SIRT7-dependent deacetylation of CDK9 activates RNA polymerase II transcription
  • Document date: 2017_3_17
    • ID: qm9urt2w_9
    Snippet: HEK293T cells expressing Flag/HA-tagged SIRT7 were lysed in buffer AM-160 (160 mM KCl, 20 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 0.2 mM EDTA, 10% glycerol, 0.5 mM DTT) supplemented with 0.1% NP-40, protease inhibitors (Roche complete) and HDAC inhibitors (500 nM TSA, 5 mM sodium butyrate, 10 mM NAM). Sequential immunoprecipitation was first performed for 4 h at 4 • C using protein G Sepharose beads coated with anti-Flag or mouse IgG as control. Af.....
    Document: HEK293T cells expressing Flag/HA-tagged SIRT7 were lysed in buffer AM-160 (160 mM KCl, 20 mM Tris-HCl [pH 7.9], 5 mM MgCl 2 , 0.2 mM EDTA, 10% glycerol, 0.5 mM DTT) supplemented with 0.1% NP-40, protease inhibitors (Roche complete) and HDAC inhibitors (500 nM TSA, 5 mM sodium butyrate, 10 mM NAM). Sequential immunoprecipitation was first performed for 4 h at 4 • C using protein G Sepharose beads coated with anti-Flag or mouse IgG as control. After elution with buffer AM-160/0.1% NP-40 supplemented with Flag peptide (400 g/ml), proteins were precipitated with anti-HA coupled to protein G Sepharose and eluted with buffer AM-250/0.2% NP-40 supplemented with HA-peptide (400 g/ml). Proteins were digested with trypsin overnight at 37 • C and tryptic peptides were analyzed by LC-MS/MS on an LTQ-Orbitrap XL mass spectrometer (Thermo Scientific) using a 2h gradient.

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