Selected article for: "ice cold PBS buffer and PBS buffer"
Author: Benharouga, Mohamed; Haardt, Martin; Kartner, Norbert; Lukacs, Gergely L.
Title: Cooh-Terminal Truncations Promote Proteasome-Dependent Degradation of Mature Cystic Fibrosis Transmembrane Conductance Regulator from Post-Golgi Compartments
  • Document date: 2001_5_28
    • ID: q3agdeju_13
    Snippet: Cells were washed with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0), containing 5 g/ml of leupeptin and pepstatin A, 10 mM iodoacetamide, and 1 mM PMSF at 4 Њ C. Nuclei and unbroken cells were removed by centrifugation (15,000 g , 15 min at 4 Њ C). Proteins were denatured in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose memb.....
    Document: Cells were washed with ice-cold PBS and lysed in RIPA buffer (150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate, pH 8.0), containing 5 g/ml of leupeptin and pepstatin A, 10 mM iodoacetamide, and 1 mM PMSF at 4 Њ C. Nuclei and unbroken cells were removed by centrifugation (15,000 g , 15 min at 4 Њ C). Proteins were denatured in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose membrane. Immunoblotting was performed as described previously, using mouse monoclonal L12B4 and M3A7 anti-CFTR antibodies (Abs; Kartner et al., 1992) , mouse monoclonal anti-HA (Covance), and antiubiquitin Abs (Santa Cruz Biotechnology, Inc.; Lukacs et al., 1994) . Primary Abs were visualized by horseradish peroxidase-conjugated sheep anti-mouse IgG and enhanced chemiluminescence (ECL Western blot kit; Amersham Pharmacia Biotech).

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