Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses.
Preparation of the Nuclear Matrix for Parallel Microscopy and Biochemical Analyses.
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用于平行显微镜和生化分析的核基质的制备。
DOI：
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发表时间：
2016
影响因子：
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通讯作者：
D. Coverley
中科院分区：
文献类型：
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作者：
Rosemary H. C. Wilson;E. Hesketh;D. Coverley
细胞核内固定的蛋白质通常通过用洗涤剂处理细胞来鉴定。耐洗涤剂的部分通常被认为是染色质，许多研究都是这样描述的。然而，这一组分由染色质结合蛋白和核基质结合蛋白组成。为了单独研究与核基质结合的蛋白质，需要进一步分离这些组分；必须去除DNA，以便将剩余的蛋白质与未经处理的细胞中的蛋白质进行比较。该方案使用非离子洗涤剂(Triton X-100)在生理浓度下去除细胞膜和可溶性蛋白质，然后用0.5M的氯化钠提取，用DNase I消化，并去除DNA片段。它使用一种特殊的缓冲器(细胞骨架缓冲器)在相对温和的条件下稳定细胞骨架和核基质。然后，可以通过免疫荧光(IF)和免疫印迹(IB)来评估核基质蛋白。IB的优点是可以分解不同形式的感兴趣蛋白质，并且可以分析可溶的部分。IF分析的主要优点是可以监测单个细胞(而不是均质的群体)，并可以揭示与残存核结构结合的蛋白质的空间排列。
Immobilized proteins within the nucleus are usually identified by treating cells with detergent. The detergent-resistant fraction is often assumed to be chromatin and is described as such in many studies. However, this fraction consists of both chromatin-bound and nuclear-matrix-bound proteins. To investigate nuclear-matrix-bound proteins alone, further separation of these fractions is required; the DNA must be removed so that the remaining proteins can be compared with those from untreated cells. This protocol uses a nonionic detergent (Triton X-100) to remove membranes and soluble proteins from cells under physiologically relevant salt concentrations, followed by extraction with 0.5 m NaCl, digestion with DNase I, and removal of fragmented DNA. It uses a specialized buffer (cytoskeletal buffer) to stabilize the cytoskeleton and nuclear matrix in relatively gentle conditions. Nuclear matrix proteins can then be assessed by either immunofluorescence (IF) and immunoblotting (IB). IB has the advantage of resolving different forms of a protein of interest, and the soluble fractions can be analyzed. The major advantage of IF analysis is that individual cells (rather than homogenized populations) can be monitored, and the spatial arrangement of proteins bound to residual nuclear structures can be revealed.