The efficient detection of membrane protein with immunoblotting: lessons from cold-temperature denaturation
Transmembrane proteins play essential roles in cell signaling, transport of membrane-impermeable molecules, cell-cell communication, and cell adhesion. Our recent work demonstrated that reactive oxygen species-generating NADPH oxidase 4 (Nox4), a protein with multiple transmembrane domains, is involved in cell migration by stabilizing vascular endothelial growth factor receptor 2 (VEGFR-2), a single-span transmembrane protein. During this study and with further verification, we developed a simple method to prepare protein samples without aggregating these membrane proteins for SDS-PAGE, immunoblotting, and deglycosylation assay. We found that heating was unnecessary for protein denaturation for SDS-PAGE and deglycosylation assay. Also, the detectable amounts of VEGFR-2 and Nox4 were increased in the sample treated at 4℃ compared with the sample treated at 98℃ . Moreover, the N -glycan of VEGFR-2 was digested by glycosidase at reaction temperature 4℃ .