In order to access full contents on LabWizards.com,
please log in or register now.
Haven't you joined our community yet? Registration is free and will grant full access to website and will allow you to
browse our protocols, upload original contents, create your own scientific portfolio and more.
Cells should be at around 90% visual confluence when harvesting. Avoid overconfluence that may result in cell detachment during the washes. Cells may be grown in 10-cm plates. For HCT116 cells, use two 10-cm plates per condition and seed 3.5 million cells in each 10-cm plate 3 days before harvesting (change medium after 2 days).
Solutions
10x Glycine solution (1.25M glycine)
SDS lysis buffer: 1%SDS, 10 mM EDTA, 50 mM Tris pH8 (prepare just before use and keep at RT. Supplement with PIC and PMSF just before use)
Dilution Buffer: 0.01% SDS, 1.1% Triton X100 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl
Low salt buffer: 0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris pH 8, 150 mM NaCl
High salt buffer: 0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris pH 8, 500 mM NaCl
LiCl buffer: 250 mM LiCl, 1% NP40, 1% NaDOC, 1 mM EDTA, 10 mM Tris pH 8
TE: 10 mM Tris, 1 mM EDTA