Selected article for: "lysis buffer and western blot"
Author: Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory
Title: Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo
  • Document date: 2018_4_6
    • ID: 1pbshnw9_7
    Snippet: Pre-miRNA-seq and miRNA-seq preparation was based on previous work (19, 29, 30) . HEK293T cells were lysed in RSB 200 (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40) supplemented with 0.2 U/μl RNasein (Promega) and protease inhibitor cocktail (Roche). Cleared lysates were incubated with Protein G agarose beads (Life Technologies) conjugated to AGO 2A8 antibody or non-immune serum (NMS), for 1.5 h at 4°C. After four 5 min washes.....
    Document: Pre-miRNA-seq and miRNA-seq preparation was based on previous work (19, 29, 30) . HEK293T cells were lysed in RSB 200 (20 mM Tris-HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl 2 , 0.5% NP-40) supplemented with 0.2 U/μl RNasein (Promega) and protease inhibitor cocktail (Roche). Cleared lysates were incubated with Protein G agarose beads (Life Technologies) conjugated to AGO 2A8 antibody or non-immune serum (NMS), for 1.5 h at 4°C. After four 5 min washes in lysis buffer, a sample of beads was drawn for western blot analysis while the rest was subjected to RNA extraction with Trizol (Life Technologies).

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