Review for Anti-MEL-CAM (MUC18 Antibody, CD146), clone A32

Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime), and total protein concentration was determined with an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime). Collected cell lysates were mixed with gel-loading buffer, equal amounts of proteins were loaded, separated on 10% SDS/PAGE, and electrophoretically transferred onto nitrocellulose membranes (Millipore), which were blocked in PBS containing Tween 20 (PBST) and 5% milk at room temperature for 2 h and blotted with Anti-MEL-CAM antibody (1:500) overnight at 4 °C. After washing with PBST, the membranes were incubated with anti-mouse horseradish peroxidase-conjugated secondary mAb (1:5,000; Zhongshan Goldenbridge) in PBST containing 5% milk, and immunocomplexes were visualized using the SuperSignal West Dura extended Duration Substrate (34076; Thermo). Images were analyzed by GeneTools image analysis software (SynGene).