determination of protein content by a bicinchoninic acid (BCA) assay

cell extracts containing 15 µg of total protein were separated on 4-15% sodium dodecyl sulfate-polyacrylamide gradient (150V, Bio-Rad) gels

electroblot gel onto polyvinylidene difluoride membrane

incubate membranes with 0.4% volume paraformaldehyde for 0h 15m 0s for better protein binding

Block membranes with 5Mass Percent milk for 0h 30m 0s

incubate blot with primary antibody for 4h 0m 0s

wash blot for 3 times for 0h 5m 0s with PBS-0.1% volume TRITON-X100

incubate for 1h 0m 0s with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup)

detect using chemiluminescence