Abstract：
Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Biochemical fractionation Support Protocol 1: Protein quantification using Bradford assay Support Protocol 2: SDS/PAGE and Western blotting.
摘要：
生化分级分离是一种用于分离和分离不同细胞区室的技术，对于解剖细胞机制和分子途径至关重要。在这里，我们概述了分离超纯细胞核和细胞质的生化部分方法。该方案利用低渗裂解缓冲液悬浮细胞，再加上校准的离心策略，用于增强细胞质与核部分的分离。随后的纯化步骤确保分离的核级分的完整性。总的来说,这种方法有助于准确的蛋白质定位，对于功能研究至关重要，证明其在分离细胞区室中的功效。©2024作者WileyPeriodicalsLLC出版的当前协议。基本方案:生化分级支持方案1:使用Bradford测定的蛋白质定量支持方案2:SDS/PAGE和Western印迹。
