Pro light chemiluminescence detection kit

Cells were lysed in NP40 lysis buffer and then cell lysates were centrifuged at 12,000 g, 4 °C for 20 min. 20 µL protein G agarose beads (Roche, 11243233001) were added to the supernatants for preclear. The supernatants were incubated with protein G agarose beads and indicated antibodies at 4 °C for 8 h. Then immunocomplexes were centrifuged at 4 °C for 3 min and NP-40 lysis buffer were used to wash the precipitates three times before subjected to western blot assay.
For western blot assay, proteins were separated on 10% or 12% SDS-PAGE gel where appropriate. Proteins were then transferred to PVDF membranes (Millipore, IPVH00010), which were blocked with 5% BSA (Genview, FA016). Next PVDF membranes were incubated with the indicated antibodies and washed 3 times with TBST (0.05% Tween-20, 150 mM NaCl and 20 mM Tris-HCl). Lastly, PVDF membranes were incubated with horseradish peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, 31460) or horseradish peroxidase-conjugated anti-mouse (Thermo Fisher Scientific, 31430) secondary antibodies. Western blot results were obtained by digital gel image analysis system (TANON 5500) and Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01).

Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Milipore). The membranes were blocked with 5% skim milk (BD, 232100) for 1 h at room temperature and incubated with the indicated antibodies overnight at 4°C and detected with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized after incubation with Pro-Light chemiluminescence detection kit (TIANGEN, PA112-01) using a digital gel image analysis system TANON 5500.