4.8. Western Blot Analyses

Sample proteins (30 μg) were diluted in 2× Laemmli buffer (Invitrogen, Carlsbad, CA, USA), heated at 70 °C for 10 min, separated on a Biorad Criterion XT 4–20% gradient Tris-glycine polyacrylamide gels (Invitrogen) by electrophoresis, and then transferred to (Polyvinylidene difluoride) PVDF membranes using the semi-dry approach (Biorad Trans-Blot^® Turbo Transfer System). Briefly, the gel was assembled in a sandwich with filtering papers and a PVDF membrane and then placed into a cassette and transferred for 7 min using a pre-set voltage (12.5 mA, 30 V). After being blocked for 1 h in 5% skim milk in TBST at room temperature, membranes were incubated overnight at 4 °C with different primary antibodies (please refer to Table 2) in 1% skim milk containing 1X TBST. Membranes were then washed and incubated with secondary antibody raised against the primary animal in 5% skim milk and TBST 1X for 1 h. The densities of the bands on the membrane were scanned and analyzed using the chemiluminescent approach on an Amersham Imager 600 System.

Primary antibodies used for Western blotting. Optimal dilutions are shown in the right column.