Summary: CD133 is widely used as a marker to isolate cancer stem cells (CSCs). However, the structural ambiguity of N-glycan of CD133 limits its application in the isolation of CSCs. Here, we present a protocol to sort CSCs from tumor samples by combining CD133 with α-1,2-high-mannose type glycan chains. We describe steps for purifying and biotinylating cyanovirin-N (CVN) proteins and sorting tumor cells and CD133+α-1,2-Man+ (α-1,2-linked mannose+) cells to provide more specific markers for CSCs.For complete details on the use and execution of this protocol, please refer to Wei et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.