Protein isolation and western blotting procedures have been described before.14 (link) Whole cell lysates from cell cultures were prepared by adding lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA, pH 8; 0.1% NP40; 0.01% SDS with protease inhibitor cocktail and phos-STOP (Roche, Germany).
For xenograft tumour lysate preparation, excised, snap frozen tumours were homogenized in above mentioned lysis buffer using Tissue Grinder, sonicated (50% amplitude for 2 min with 10 s on and 20 s off pulses) and lysates were cleared by centrifugation.
Total proteins isolated were quantified by Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of lysates were resolved in appropriate SDS-PAGE gels, transferred onto 0.45 μm PVDF membrane (Millipore, Banglore, India) and probed with primary antibodies. HRP-conjugated secondary antibodies and Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) were used for protein band detection using GeneGnome system (Syngene, Cambridge, UK). Anti-GAPDH alone or in combination with anti-histone-H3 antibody was used as protein loading and transfer control.
For xenograft tumour lysate preparation, excised, snap frozen tumours were homogenized in above mentioned lysis buffer using Tissue Grinder, sonicated (50% amplitude for 2 min with 10 s on and 20 s off pulses) and lysates were cleared by centrifugation.
Total proteins isolated were quantified by Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of lysates were resolved in appropriate SDS-PAGE gels, transferred onto 0.45 μm PVDF membrane (Millipore, Banglore, India) and probed with primary antibodies. HRP-conjugated secondary antibodies and Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) were used for protein band detection using GeneGnome system (Syngene, Cambridge, UK). Anti-GAPDH alone or in combination with anti-histone-H3 antibody was used as protein loading and transfer control.
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