Protocol for high-sensitivity western blot on murine hematopoietic stem cells

Abstract

Summary: Hematopoietic stem cells (HSCs) sustain hematopoiesis during homeostasis and regeneration. However, their limited availability poses a challenge for protein analysis. Here, we present a protocol for performing high-sensitivity western blot on HSCs using two techniques that enhance HSC isolation from mice and boost sensitivity for low cell numbers. We describe steps for isolating murine bone marrow cells, antibody staining, and cell sorting and post-sort analysis. We then detail a western blot procedure suitable for low numbers of HSCs.For complete details on the use and execution of this protocol, please refer to Li et al (2022).1,2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Isolation
• Flow Cytometry/Mass Cytometry
• Protein Biochemistry
• Stem Cells