Suzuki Keiko, Bose Pinaki, Leong-Quong Rebecca Yy, Fujita Donald J, Riabowol Karl
Department of Biochemistry & Molecular Biology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, T2N 4N1, Canada.
BMC Res Notes. 2010 Nov 10;3:294. doi: 10.1186/1756-0500-3-294.
The translocation or shuttling of proteins between the nucleus and cytoplasm (nucleocytoplasmic transport [NCPT]) is often a rapid event following stimulation with growth factors or in response to stress or other experimental manipulations. 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. 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. 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.
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.
蛋白质在细胞核与细胞质之间的转运或穿梭（核质转运 [NCPT]）通常是在生长因子刺激后、对应激或其他实验操作作出反应时的快速事件。常用的从细胞质中分离细胞核的方法采用冗长的步骤，如密度梯度离心，这会使细胞长时间暴露于非生理性高渗条件下，导致细胞核与细胞质之间出现不同程度的渗漏。为了帮助维持和量化蛋白质的核质比，已使用诸如 leptomycin B 等试剂，以便通过抑制核输出更好地分析 NCPT。为了在不存在可能引入未知假象的这些实验操作的情况下追踪 NCPT，我们开发了一种快速方法，该方法似乎能产生纯的细胞核和细胞质组分，适用于准确估计已知经历 NCPT 的蛋白质的核质比。
我们开发了一种用于培养的原代和转化人细胞亚细胞分级分离的快速、高效且实用（REAP）方法。REAP 方法是一种基于非离子去污剂的两分钟纯化技术，仅需要台式离心机、微量移液器和微量离心管。这种廉价的方法已证明能有效分离细胞核蛋白和细胞质蛋白，这通过核孔蛋白和核纤层蛋白 A 核标记物或丙酮酸激酶和微管蛋白细胞质标记物无可检测到交叉污染来估计。REAP 组分也反映了通过间接免疫荧光平行观察到的 TNFα 诱导的 NF-κB NCPT。
该方法极大地减少了亚细胞分级分离所需的时间，消除了可检测到的蛋白质降解并维持了蛋白质相互作用。该程序的简单性、简短性和效率允许在维持蛋白质完整性和蛋白质复合物相互作用的同时追踪蛋白质亚细胞重新定位中的短暂变化。
