Polysomal Profiling of Cell Lysates
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Corresponding Organization :
Other organizations : University of Trento, University of Milan, University College London, National Hospital for Neurology and Neurosurgery, UK Dementia Research Institute, New York University, IRCCS Istituto Auxologico Italiano, Istituti di Ricovero e Cura a Carattere Scientifico
Variable analysis
- Treatment with CHX (10 µg/ml) for 4 min
- Polysomal profiling
- Cell seeding in 10-cm dishes
- Lysis buffer composition [10 mM NaCl, 10 mM MgCl2•6H2O, 10 mM Tris–HCl, pH 7.5, 1% Triton X-100, 0.2 U/μl Ribolock RNase inhibitor, 0.0005 U/μl DNaseI, CHX 10 μg/ml, 1 mM dithio-threitol, 1% sodium deoxycholate]
- Centrifugation of lysate at 1,620 × g for 5 min to pellet cell debris
- Sucrose gradient composition (10–40% [w/v])
- Centrifugation of lysate on sucrose gradient at 187,813 × g for 1 h 30 min
- Fraction collection monitoring absorbance at 254 nm
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