Proteins in sample buffer were heated at 37°C, resolved by SDS-polyacrylamide gel electrophoresis in 10% acrylamide gels using tricine buffer, and transferred to nitrocellulose membranes.
Samples (50 µg protein in 30-µl Laemmli buffer) were heated at 95°C for 5 min, resolved by 10% SDS-PAGE (50 µg/lane), and electro-transferred to PVDF.
Cell lysates in SDS-PAGE sample buffer were heated at 95°C for 5 min, separated by 12.5% SDS-PAGE, and transferred to a nitrocellulose membrane.
The PCR conditions were as follows: 1 cycle at 94°C for 5 min; 35 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and 1 cycle at 72°C for 10 min. PCR products mixed with loading buffer were heated at 95°C for 5 min and quickly chilled on ice.
One μg of each labeled cRNA was fragmented by adding 5 μl 10 × blocking agent and 1 μl of 25 × fragmentation buffer, then the mixture was heated at 60°C for 30 minutes; finally, 25 μl 2 × GE hybridization buffer was added to dilute the labeled cRNA.
The slides were immersed in 0.01 M sodium citrate buffer (pH 6.0), which was heated at 95°C for 15 minutes.
The immune complex was eluted twice with 250 μl of elution buffer and the eluate was heated at 65°C for at least 6 h to reverse the cross-linking.
One μg of each labeled cRNA was fragmented by adding 5 μl 10 × blocking agent and 1 μl of 25 × fragmentation buffer, after which the mixture was heated at 60°C for 30 minutes, and 25 μl 2 × GE hybridization buffer was added to dilute the labeled cRNA.
Following the final wash, 40 µl of SDS gel-loading buffer was added, the mixture was heated at 100°C for 3 min, and proteins were resolved by SDS-PAGE.
