REAP: A two minute cell fractionation method.

Area of Science:

• Cell Biology
• Molecular Biology

Background:

• Nucleocytoplasmic transport (NCT) is crucial for cellular function and often rapid.
• Traditional methods for studying NCT involve lengthy procedures that can introduce artifacts.
• Existing methods may compromise nuclear:cytoplasmic ratios and protein integrity.

Purpose of the Study:

• To develop a rapid and artifact-free method for subcellular fractionation.
• To enable accurate quantification of nuclear:cytoplasmic protein ratios.
• To facilitate the study of dynamic changes in nucleocytoplasmic transport.

Main Methods:

• Developed a Rapid, Efficient And Practical (REAP) method.
• Utilized a two-minute, non-ionic detergent-based purification technique.
• Employed standard laboratory equipment: tabletop centrifuge, micro-pipette, micro-centrifuge tubes.

Main Results:

• Achieved pure nuclear and cytoplasmic fractions in primary and transformed human cells.
• Demonstrated no detectable cross-contamination of nuclear (nucleoporin, lamin A) or cytoplasmic (pyruvate kinase, tubulin) markers.
• Observed results consistent with TNFα-induced NF-κB nucleocytoplasmic transport (NCPT) seen via immunofluorescence.

Conclusions:

• The REAP method significantly reduces fractionation time.
• Eliminates detectable protein degradation and preserves protein interactions.
• Enables tracking of rapid subcellular protein relocalization while maintaining integrity and complex interactions.