REAP: A two minute cell fractionation method

We have developed a Rapid, Efficient And Practical (REAP) method for subcellular fractionation of primary and transformed human cells in culture. 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. 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. REAP fractions also mirrored TNFα induced NF-κB NCPT observed in parallel by indirect immunofluorescence.This method drastically reduces the time needed for subcellular fractionation, eliminates detectable protein degradation and maintains protein interactions. 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.Subcellular fractionation was first described by Albert Claude in 1946 [1,2]. He wrote: "The physiology of the cell cannot be fully understood unless we succeed in determining the constitution of its parts,..." [2]. 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 [3]. Christian de Duve pioneered the use of sucrose density gradients to fractionate cells in 1951 [4,5] and subsequent researchers have developed various additional modifications [6-8]. 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. The nucleus and the cytoplasm have very distinct macromolecular composition and separation of nuclear and cytosolic fractions is proving very useful for proteomic analysis [9]. A majority of the establi