{"title":"Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis","authors":"Hang T. Huynh, Evgeniia Shcherbinina, Hsiang-Chi Huang, Reyhaneh Rezaei, Aishe A. Sarshad","doi":"10.1002/cpz1.1042","DOIUrl":null,"url":null,"abstract":"<p>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.</p><p><b>Basic Protocol</b>: Biochemical fractionation</p><p><b>Support Protocol 1</b>: Protein quantification using Bradford assay</p><p><b>Support Protocol 2</b>: SDS/PAGE and Western blotting</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cpz1.1042","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.1042","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
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
用于下游分子分析的细胞质和核馏分的生化分离。
生化分馏是一种用于分离不同细胞区室的技术,对于剖析细胞机制和分子途径至关重要。在此,我们概述了一种用于分离超纯细胞核和细胞质的生化分馏方法。该方案利用低渗裂解缓冲液使细胞悬浮,并采用校准离心策略,以加强细胞质与核部分的分离。随后的纯化步骤可确保分离出的核部分的完整性。总之,这种方法有助于准确定位蛋白质,对功能研究至关重要,证明了它在分离细胞区室方面的功效。© 2024 作者。当前协议》由 Wiley Periodicals LLC 出版。基本方案:支持方案 1:使用 Bradford 检测法进行蛋白质定量 支持方案 2:SDS/PAGE 和 Western 印迹。
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