Single cell-resolution western blotting.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Conventional western blotting faces limitations with specific protein targets and background noise.
• Measuring cell-to-cell variation in protein expression requires single-cell resolution.

Purpose of the Study:

• To describe a protocol for single-cell western blotting (scWB).
• To enable measurement of cell-to-cell variation in protein levels and states.
• To provide a method applicable to protein targets lacking selective antibodies.

Main Methods:

• scWB utilizes a microdevice with microwells in a polyacrylamide gel (PAG).
• The protocol involves cell settling, lysis, PAGE, UV-induced blotting, and in-gel immunoprobing.
• Multiplexing up to 10+ proteins per cell is achievable via antibody cocktails and stripping/reprobing.

Main Results:

• The scWB protocol allows for high-selectivity, multiplexed protein detection in single cells.
• The assay can be completed in 4-6 hours by novices.
• Microdevice fabrication for uniform and pore-gradient microgels is described, along with optional de-cross-linking for small pore-size gels.

Conclusions:

• scWB is a powerful technique for direct protein measurement at the single-cell level.
• It is relevant for fundamental biosciences and applied biomedicine.
• The method addresses limitations of conventional western blotting for specific applications.