染色质免疫沉淀技术在研究DNA与蛋白质相互作用中的应用

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域。与其他方法相比,染色质免疫沉淀技术（chromatin immunoprecipitation assay, ChIP）是一种在体内研究DNA-蛋白质相互作用的理想的方法。近年来这种方法与DNA芯片和分子克隆技术相结合,可用于高通量的筛选已知蛋白因子的未知DNA靶点和研究反式作用因子在整个基因组上的分布情况,这将有助于深入理解DNA-蛋白质相互作用的调控网络。总结了染色质免疫沉淀技术的方法,特别介绍了使用这些方法取得的最新进展。     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

Bi-functional cross-linking reagents efficiently capture protein-DNA complexes in Drosophila embryos

Chromatin immunoprecipitation (ChIP) is widely used for mapping DNA-protein interactions across eukaryotic genomes in cells, tissues or even whole organisms. Critical to this procedure is the efficient cross-linking of chromatin-associated proteins to DNA sequences that are in close proximity. Since the mid-nineties formaldehyde fixation has been the method of choice. However, some protein-DNA complexes cannot be successfully captured for ChIP using formaldehyde. One such formaldehyde refractory complex is the developmentally regulated insulator factor, Elba. Here we describe a new embryo fixation procedure using the bi-functional cross-linking reagents DSG (disuccinimidyl glutarate) and DSP (dithiobis[succinimidyl propionate). We show that unlike standard formaldehyde fixation protocols, it is possible to capture Elba association with insulator elements in 2–5 h embryos using this new cross-linking procedure. We show that this new cross-linking procedure can also be applied to localize nuclear proteins that are amenable to ChIP using standard formaldehyde cross-linking protocols, and that in the cases tested the enrichment was generally superior to that achieved using formaldehyde cross-linking.     

染色质免疫共沉淀实验方法

染色质免疫共沉淀(ChIP)技术是一种检测蛋白质与DNA结合的实验技术。该方法可以先进行样品交联, 然后将蛋白质与DNA复合物进行随机DNA切断, 再借助免疫学方法特异性富集与目的蛋白相结合的DNA片段, 从而检测转录因子等目的蛋白质与DNA的结合情况, 鉴定基因启动子或其它DNA结合位点。该方法同时也可应用于研究基因组特定位点的组蛋白修饰情况。该文介绍了依赖交联固定的常规免疫共沉淀(X-ChIP), 以及适用于10³细胞级别微量实验材料的基于微球菌核酸酶非交联免疫共沉淀(ULI-NChIP)具体操作过程和注意事项。     

Southwestern blotting in investigating transcriptional regulation

Southwestern blotting is used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA footprinting is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques; a denaturing SDS-PAGE is first employed to separate proteins electrophoretically based on size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labeled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein(s) is later visualized by autoradiography. This technique could be combined with database searching (TransFac, http://www.gene-regulation.com/pub/databases.html#transfac), prediction of potential protein factors binding onto a target motif (e.g., Patch search), in vitro supershift EMSA and in vivo chromatin immunoprecipitation (ChIP) assays for effective identification of protein factors. The whole Southwestern blotting procedure takes approximately 4 d to complete. In this article, a commonly used protocol and expected results are described and discussed.     

Reverse ChIP:研究DNA-蛋白质相互作用的新方法

反向染色质免疫共沉淀技术(reverse chromatin immunoprecipitation assay,Reverse ChIP)是一种在体内状态下分析DNA-蛋白质相互作用的新方法.它用特异的核酸探针捕获靶DNA片段及与其相结合的蛋白质,蛋白质用质谱仪检测,以达到确定靶DNA位点全部相关蛋白质的目的.其可对靶DNA位点相关蛋白质进行全面、系统地鉴定,特别是寻找已知DNA元件相应的调节蛋白.在发现、鉴定靶DNA位点相关蛋白质和研究DNA-蛋白质相互作用中有重要应用价值.     

ChIP技术及其在基因组水平上分析DNA与蛋白质相互作用

染色质免疫沉淀(Chromatin immunoprecipitaion, ChIP)技术是分析细胞内生理状态下DNA结合蛋白与基因组DNA相互作用的技术。ChIP与高密度芯片(ChIP-chip)或高通量测序(ChIP-Seq)相结合能产生大量的研究数据, 在细胞的基因表达调控网络研究中发挥重要作用。文章主要介绍ChIP、ChIP-chip和ChIP-Seq的技术特点以及发展趋势, 重点讨论了ChIP-Seq数据分析方法及相关的应用实例。     

BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.     

运用染色质免疫沉淀技术筛选出AP-2α的靶基因GALK1

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.与其他方法相比,染色质免疫沉淀技术 (chromatin immunoprecipitation assay, ChIP ) 是一种近来研究体内 DNA 与蛋白质相互作用的最好方法之一.本研究利用 ChIP 克隆方法, 找出了 AP-2α所调控的新的下游靶基因 GALK1,并应用 Luciferase assay和 RT-PCR 实验进行了初步的验证.这一新发现,有利于我们进一步研究转录因子AP-2α的功能.     

Fast chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) is a widely used method to explore in vivo interactions between proteins and DNA. The ChIP assay takes several days to complete, involves several tube transfers and uses either phenol–chlorophorm or spin columns to purify DNA. The traditional ChIP method becomes a challenge when handling multiple samples. We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA. This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.