3.6. Western Blotting

In this assay, the 12 h and 24 h of culture duration was predetermined for the MAPK pathway assay and 7 days of culture duration was predetermined for the osteogenic-related protein. Firstly, cells were washed thrice with PBS and lysed with NP40 (Thermo Fisher, Waltham, MA, USA). The total protein concentrations were measuring using Quick StartTM Bradford protein assay kit (Bio-Rad, Hercules, CA, USA). Sample buffer was added and the solution was placed in a dry bath at 95 °C for 10 min to allow denaturation of the protein. Cell lysates of 40 μg protein/sample were segregated using the sodium dodecyl sulfate-polyacrylamide (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) that was prepared according to the kDa of the target protein antibody. After the end of the electrophoresis, the protein was transferred to a PVDF membrane and blocked with 5% BSA for 1 h. Antibody concentration was recommended by referring to the datasheet to add the diluted primary antibody—ERK, pERK, p38, pp38, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC) from GeneTex (San Antonio, TX, USA). It was then placed on a 4 °C shaker and incubated overnight. The PVDF membrane was washed 3 times with TBST for 5 to 10 min each, then the secondary antibody was added and incubated for 1 h at room temperature. Protein bands were visualized by Vilber FUSION SOLO image scanning to detect the protein expression. Using β-actin (GeneTex) as an internal reference protein, the test was repeated in triplicate.