Nupage bis tris gradient sds page gels

Cell pellets were lysed by incubation at 4°C for 10 min in IP Lysis buffer (Pierce). Proteins were collected by centrifugation. Approximately 40 μg protein was separated on 6% to 12% NuPAGE Bis-Tris gradient SDS-PAGE gels (Life Technologies), and transferred onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in 1×PBS-T at room temperature for 30 min. Membranes were then incubated with primary antibodies (ADAR1-ab126745; GAPDH- Santa Cruz 32,233) at a 1:1,000 dilution at room temperature for 2 h. After three washes with 1×PBS-T, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad 1706515 or 1706516) at room temperature for 1 h to develop the image using Immobilon Forte Western HRP Substrate (Millipore).

Cells were collected using trypsin, and cell pellets were lysed by incubation for 30 min at 4°C in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 1% Triton X-100 and 0.5% sodium deoxycholate). Proteins were cleared by centrifugation. Approximately 20–40 μg protein were separated on 6–12% NuPAGE Bis–Tris gradient SDS-PAGE gels (Life Technologies) and transferred onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in 1× PBS at room temperature for 30 min. Membranes were then incubated with primary antibodies (1:1000–1:2000) in 5% milk at room temperature for 2 h or at 4°C overnight. After three washes with 1 × PBS, 5 min each, the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 h. After three washes with 1× PBS, images were developed using Immobilon Forte Western HRP Substrate (Millipore) and visualized using ChemiDoc system (Bio-Rad). Antibodies for RNase H1 and RNase H2A were raised in Rabbit, as described previously (30 (link),31 (link)).

Cell pellets were lysed by incubation at 4°C for 30 min in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.5 mM EDTA). Proteins were collected by centrifugation. Approximately 20–40 μg protein were separated on 6–12% NuPAGE Bis-Tris gradient SDS-PAGE gels (Life Technologies), and transferred onto PVDF membranes using the iBLOT transfer system (Life Technologies). The membranes were blocked with 5% non-fat dry milk in 1 × PBS at room temperature for 30 min. Membranes were then incubated with primary antibodies at room temperature for 2 h or at 4°C overnight. After three washes of 5 min each with wash buffer (0.05% Tween-20 1× PBS), the membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:2000) at room temperature for 1 h. After three washes with wash buffer, images were developed using Immobilon Forte Western HRP Substrate (Millipore), and visualized using ChemiDoc system (Bio-Rad).