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强烈推介:1天内完成实验的快速chip试剂盒

一、Chip实验介绍
染色质免疫沉淀技术(Chromatin Immunoprecipitation,简称ChIP)是研究体内蛋白质与DNA相互作用的一种技术。其原理是:在生理状态下用甲醛把细胞内的DNA与 蛋白质交联在一起,通过超声或酶处理将染色质切为均一的小片段后,利用抗原抗体的特异性识别反应,使用DNA结合蛋白特异性的抗体将目的蛋白和DNA复合 物沉淀下来。复合物经过洗涤,解交联,蛋白酶K消化去除DNA结合蛋白,经纯化得到感兴趣的DNA片段。纯化的DNA片段可用于后续的分析,比如 qPCR,测序,ChIP-chip,ChIP re ChIP等。
 
二、强烈推荐Active motif公司的ChIP-IT® Express试剂盒
1. Active motif公司的ChIP-IT® Express试剂盒使用protein G包被的磁珠,代替传统的琼脂糖珠,使得ChIP实验可以在1天之内完成。试剂盒可以使用超声法或酶切法进行染色质的断裂处理。每种方法都提供快速、可重复的操作步骤,省时省力,实验者更容易获得成功的ChIP结果。
 
 
图1 ChIP-IT® Express试剂盒的操作流程
 
 
2. ChIP-IT® Express试剂盒的优点
  ●  组分完整——提供ChIP实验所需的所有试剂;
  ●  可用于检测蛋白/DNA的相互作用,不仅仅限于组蛋白的修饰;
  ●  与全基因组分析或PCR相关的方法兼容;
  ●  无需进行试剂和操作步骤的优化;
  ●  无需酚/氯仿抽提步骤——试剂盒包含DNA纯化柱,可对DNA进行快速纯化
 
 
3. Active motif公司的ChIP-IT® Express试剂盒与传统试剂盒的对比
  1) 传统的ChIP实验都是使用染色质组成相关的抗体,如乙酰化组蛋白抗体。然而对于启动子转录活性分析而言,这无法揭示出是哪种转录因子结合到我们感兴趣的启 动子上。相比之下,使用转录因子特异性抗体,能够直接监测转录因子和DNA的相互作用,但是这种技术与传统的ChIP相比具有更大的挑战,需要准备多种复 杂的缓冲液,抑制剂混合物和封闭试剂等。而且在缺少抗体、对照和验证的实验流程下,很难对ChIP结果进行验证。为了解决这些问题,使得ChIP实验同时 适用于转录因子和组蛋白,Active motif公司的ChIP-IT® Express试剂盒提供ChIP实验所需的所有试剂,包括阳性对照和阴性对照,使得研究者更容易成功的操作ChIP。
 
  2) PCR分析ChIP后的DNA。典型的ChIP实验每次需要2000,000个细胞,使用ChIP-IT Express试剂盒,仅需要100,000或更少的细胞既可获得阳性的ChIP结果。
用1%的甲醛固定Hela细胞10分钟,使用超声法破碎染色质,使用阴性对照IgG和RNA pol II抗体,对不同细胞数量来源(100,000到750,000个细胞)的染色质进行ChIP,重复2次。使用阳性对照引物GAPDH对ChIP后的DNA进行36个循环的PCR分析(抗体和引物包含在人ChIP-IT对照试剂盒里,Active motif也可以提供小鼠和大鼠对照试剂盒)。取10ul PCR产物进行1%凝胶电泳,EB染色后紫外观察。结果显示与使用阴性对照IgG相比,使用RNA pol II抗体的ChIP大量富集了GAPDH启动子区域的DNA序列。
图2: 改良的ChIP(使用ChIP-IT Express试剂盒)

三、Chip试剂盒具体产品介绍


 
四、近年来,部分ChIP-IT® 试剂盒的文献介绍
1. Express (Catalog #53008)
“Sulforaphane reactivates cellular antioxidant defense by inducing Nrf2/ARE/Prdx6 activity during aging and oxidative stress.” by Kubo et al. (2017) Scientific Reports 7(14130): 1-17.
“cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.” by Kerur et al. (2018) Nature Medicine 24: 50-61.
“An integrated transcriptome and epigenome analysis identifies a novel candidate gene for pancreatic cancer.” by Jia et al. (2013) BMC Med Genomics 6(33).
“CXCL12 protects pancreatic β-cells from oxidative stress by a Nrf2-induced increase in catalase expression and activity.” by Dinić et al. (2016) Proc Jpn Acad Ser B Phys Biol Sci. 92(9): 436-454.
“Novel computational analysis of protein binding array data identifies direct targets of Nkx2.2 in the pancreas.” by Hill et al. (2011) BMC Bioinformatics 12(62).
“PARP-1 and YY1 are important novel regulators of CXCL12 gene transcription in rat pancreatic beta cells.” by Marković et al. (2013) PLoS ONE 8(3): e59679.
“Synergistic activations of REG I α and REG I β promoters by IL-6 and Glucocorticoids through JAK/STAT pathway in human pancreatic β cells.” by Yamauchi et al. (2015) J Diabetes Res. Epub: 173058.
“Localization of Double-Strand Break Repair Proteins to Viral Replication Compartments following Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus.” by Hollingworth et al. (2017) J Virol. 91(22): e00930-17.
“Crosstalk between histone modifications indicates that inhibition of arginine methyltransferase CARM1 activity reverses HIV latency.” by Zhang et al. (2017) Nucleic Acids Res 45(16): 9348-9360.
“The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2.” by DeSmet et al. (2016) PLoS Pathogens 12(10): e1005934.
“Distinctive patterns of epigenetic marks are associated with promoter regions of mouse LINE-1 and LTR retrotransposons.” by Rangasamy. (2013) Mob DNA 4(1):27.
“Phosphorylation State of ZFP24 Controls Oligodendrocyte Differentiation.” by Elbaz et al. (2018) Cell Rep 23(8):2254-2263.
“Changes in chromatin state reveal ARNT2 at a node of a tumorigenic transcription factor signature driving glioblastoma cell aggressiveness.” by Bogeas et al. (2018) Acta Neuropathol 135(2):267-283.
“Polymorphism in Tmem132d regulates expression and anxiety-related behavior through binding of RNA polymerase II complex.” by Naik et al. (2018) Transl Psychiatry 8(1):1.
“MELK is a novel therapeutic target in high-risk neuroblastoma.” by Guan et al. (2018) Oncotarget 9(2): 2591–2602.
“The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma.” by Kijewska et al. (2018) Oncotarget 8(10):16340-16355.
“BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells.” by Ciechomska et al. (2016) Scientific Reports 6(38723).
“Enhancing dopaminergic signaling and histone acetylation promotes long-term rescue of deficient fear extinction.” by Whittle et al. (2016) Transl Psychiatry 6(12):e974.
“Tissue mechanics promote IDH1-dependent HIF1α-tenascin C feedback to regulate glioblastoma aggression.” by Miroshnikova et al. (2016) Nat Cell Bio 18(12):1336-1345.

2. ChIP-IT® Express Enzymatic (Catalog #53009)
“Polymorphism rs7278468 is associated with Age-related cataract through decreasing transcriptional activity of the CRYAA promoter.” by Ma et al. (2016) Scientific Reports 6(23206).
“Deep sequencing and in silico analyses identify MYB-regulated gene networks and signaling pathways in pancreatic cancer.” by Azim et al. (2016) Sci. Rep. 6(28446).
“Overexpression of IFN-induced protein with tetratricopeptide repeats 3 (IFIT3) in pancreatic cancer: cellular "pseudoinflammation" contributing to an aggressive phenotype.” by Niess et al. (2015) Oncotarget 6(5): 3306-3318.
“Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro.” by Murai et al. (2018) Int J Mol Sci. 19(2): E108.
“LIMD1 is induced by and required for LMP1 signaling, and protects EBV-transformed cells from DNA damage-induced cell death.” by Wang et al. (2018) Oncotarget 9(5): 6282–6297.
“Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.” by Lafferty et al. (2017) Viruses 9(5): E111.
“CCCTC-binding factor recruitment to the early region of the human papillomavirus 18 genome regulates viral oncogene expression.” by Paris et al. (2015) J Virol. 89(9):4770-85.
“Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans.” by Zahn et al. (2015) Genome Biol 16:74.
“HIV-1 induced nuclear factor I-B (NF-IB) expression negatively regulates HIV-1 replication through interaction with the long terminal repeat region.” by Vemula et al. (2015) Viruses 7(2):543-58.
“Inflammation-induced miRNA-155 inhibits self-renewal of neural stem cells via suppression of CCAAT/enhancer binding protein β (C/EBPβ) expression.” by Obora et al. (2017) Sci Rep 7:43604.
“BH3 mimetics suppress CXCL12 expression in human malignant peripheral nerve sheath tumor cells.” by Graham et al. (2017) Oncotarget 8(5): 8670–8678.
 
3. ChIP-IT® Protein G Magnetic Beads (included in Catalog #53008 & #53009)
“RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation.” by Ferrai et al. (2017) Mol Syst Biol 13(10):946.
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