One-step extraction of subcellular proteins from eukaryotic cells.

Area of Science:

• Biochemistry
• Cell Biology
• Bioanalytical Chemistry

Background:

• Subcellular protein localization is crucial for cellular function.
• Conventional methods for analyzing subcellular proteins require complex, multi-step procedures like cell lysis and centrifugation.
• These traditional techniques are often incompatible with small or scarce cell samples.

Purpose of the Study:

• To develop a simplified, single-step method for selective extraction of cytosolic proteins.
• To overcome the limitations of conventional subcellular fractionation techniques.
• To enable efficient spatial proteomics and monitoring of protein translocation.

Main Methods:

• Application of microfluidic flow-through electroporation for cell membrane permeabilization.
• Selective extraction of cytosolic proteins in a single step.
• Monitoring of transcription factor NF-kappaB translocation as a proof of concept.

Main Results:

• Successfully demonstrated a single-step method for breaching cell membranes and extracting cytosolic proteins.
• Showcased the ability to monitor NF-kappaB translocation from cytosol to nucleus without subcellular fractionation.
• Validated compatibility with various sample sizes, from scarce to larger volumes.

Conclusions:

• Microfluidic flow-through electroporation offers a simplified and universal tool for bioanalytical analysis.
• The technique eliminates the need for bulky equipment and complex fractionation steps.
• Enables advanced spatial proteomics and real-time monitoring of protein dynamics.