A majority of the established methods of subcellular fractionation are based on subtle variations of the sucrose density gradient method, often with addition of detergents to solubilize membrane proteins. The nucleus and the cytoplasm have very distinct macromolecular composition and separation of nuclear and cytosolic fractions is proving very useful for proteomic analysis. Over the last 60-70 years, cell fractionation has provided biologists with valuable reagents to provide insight into cellular architecture, composition and function of cellular organelles. Christian de Duve pioneered the use of sucrose density gradients to fractionate cells in 1951 and subsequent researchers have developed various additional modifications. Subsequently, Claude's method was improved upon by Hogeboom, Schnieder and Palade to obtain the nuclear fraction which was discarded in Claude's original method along with cell debris. He wrote: "The physiology of the cell cannot be fully understood unless we succeed in determining the constitution of its parts.". Subcellular fractionation was first described by Albert Claude in 1946. The simplicity, brevity and efficiency of this procedure allows for tracking ephemeral changes in subcellular relocalization of proteins while maintaining protein integrity and protein complex interactions. This method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence. This inexpensive method has proven to efficiently separate nuclear from cytoplasmic proteins as estimated by no detectible cross-contamination of the nucleoporin and lamin A nuclear markers or the pyruvate kinase and tubulin cytoplasmic markers. The REAP method is a two minute non-ionic detergent-based purification technique requiring only a table top centrifuge, micro-pipette and micro-centrifuge tubes. We have developed a R apid, E fficient A nd P ractical ( REAP) method for subcellular fractionation of primary and transformed human cells in culture. To track NCPT in the absence of these experimental manipulations that could introduce unknown artefacts, we have developed a rapid method that appears to produce pure nuclear and cytoplasmic fractions, suitable for obtaining accurate estimates of the nuclear:cytoplasmic ratios of proteins known to undergo NCPT. To help maintain and quantify nuclear:cytoplasmic ratios of proteins, agents such as leptomycin B have been employed to be able to better analyze NCPT by inhibiting nuclear export. Commonly used methods to separate nuclei from cytoplasm employ lengthy steps such as density gradient centrifugation which exposes cells to non-physiological hyperosmotic conditions for extended time periods resulting in varying degrees of leakage between the nucleus and cytoplasm. The translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport ) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations.
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