ChIP实验精讲（做科研的必看）(3)

RNA. 2. Preparation of mRNA-free protein synthesissystem from rabbit reticulocyte lysates. 3. Study of chromatin structure.

23. ChIP试剂盒适用于细菌么？

答：细菌应该也能用。之前有两篇论文可以做参考：

1. Molecular basis for the exploitation of sporeformation as survival mechanism by virulent

phage phi29. WJ Meijer, V Castilla-Llorente, L Villar, HMurray, J Errington, and M Salas

EMBO J, October 19, 2005; 24(20): 3647-57.

http://highwire.stanford.edu/cgi/medline/pmid;16193065?maxtoshow=&HITS=&hits=&RESUL

TFORMAT=1&author1=Meijer&andorexacttitle=and&fulltext=molecular+basis+for+the+exploit

ation+of+spore+formation+&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=r

elevance&resourcetype=HWCIT

2. Identification of TonB homologs in the familyEnterobacteriaceae and evidence for

conservation of TonB-dependent energy transduction complexes

RA Larsen, PS Myers, JT Skare, CL Seachord, RP Darveau, andK Postle J. Bacteriol., Mar 1996; 178: 1363 - 1373.

http://highwire.stanford.edu/cgi/searchresults?fulltext=Identification+of+TonB+Homologs&and

orexactfulltext=and&searchsubmit=redo&resourcetype=1&search=Search&author1=Larsen&

pubdate_year=1996&volume=&firstpage=&src=ml

24. 我能使用ChIP试剂盒做组织样品么？

答：可以。ChIP试剂盒已被成功用于肝脏、脾脏、结肠和全鼠胚胎。

25. Q: EZ ChIP试剂盒和常规ChIP试剂盒之间的区别是什么？

答： 关键区别在于DNA的纯化方式，EZChIP用纯化柱纯化DNA。

26. 进行IgG纯化时蛋白质A和蛋白质G的区别是？

答： 蛋白质A结合到IgG的Fc部分。蛋白质G选择性结合到IgG的Fc部分，但也能

结合到Fab区域，因此可用于纯化IgG1的Fab片段。 21. Protein L与ProteinA, Protein G的区别

答：ProteinL is an immunoglobulin-binding protein that originates from the

bacteriaPeptostreptococcus magnus. Unlike Protein A and Protein G, which bind primarilythrough Fc regions (i.e., heavy chain) of immunoglobilins, Protein L binds Igsthrough interactions with the light chains. Since no part of the heavy chain isinvolved in the binding interaction, Protein L binds a wider range of Igclasses than Protein A or G. Protein L binds to representatives of all classesof Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments(ScFv) and Fab fragments also bind to Protein L. Fromhttp://www.piercenet.com/Objects ... ctFamilyID=01010323

Despite this wide-ranging binding capability with respect toIg classes, Protein L is not a universal immunoglobilin-binding protein.Binding of Protein L to immunoglobulins is restricted to those containing kappalight chains (i.e., k chain of the VL domain).1 In humans and mice, kappa (k)light chains predominate. The remaining immunoglobulins have lambda (l) lightchains. Furthermore, Protein L is effective in binding only certain subtypes ofkappa light chains. For example, it binds human VkI, VkIII and VkIV subtypesbut does not bind the VkII subtype. Binding of mouse immunoglobulins isrestricted to those having VkI light chains.

22. 关于酶切DNA片段，Micrococcal Nuclease Enzyme是什么？

答：Itpreferentially digests single-stranded DNA and AT or AU-rich regions but isalso active against RNA and double-stranded DNA (all sequences are ultimatelycleavable). Products of digestion are nucleic acid fragments containing 3'phosphate termini. Exhaustive digestion with excess enzyme yields mono- anddinucleotides. The enzyme requires calcium ions for activity and is easilyinactivated by EDTA or EGTA.

Application. 1. Removal of nucleic acids, especiallysingle-stranded DNA or RNA. 2. Preparation of mRNA-free protein synthesissystem from rabbit reticulocyte lysates. 3. Study of chromatin structure.

23. ChIP试剂盒适用于细菌么？

答：细菌应该也能用。之前有两篇论文可以做参考：

1. Molecular basis for the exploitation of sporeformation as survival mechanism by virulent

phage phi29. WJ Meijer, V Castilla-Llorente, L Villar, HMurray, J Errington, and M Salas

EMBO J, October 19, 2005; 24(20): 3647-57.

http://highwire.stanford.edu/cgi/medline/pmid;16193065?maxtoshow=&HITS=&hits=&RESUL

TFORMAT=1&author1=Meijer&andorexacttitle=and&fulltext=molecular+basis+for+the+exploit

ation+of+spore+formation+&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=r

elevance&resourcetype=HWCIT

2. Identification of TonB homologs in the familyEnterobacteriaceae and evidence for

conservation of TonB-dependent energy transduction complexes

RA Larsen, PS Myers, JT Skare, CL Seachord, RP Darveau, andK Postle J. Bacteriol., Mar 1996; 178: 1363 - 1373.

http://highwire.stanford.edu/cgi/searchresults?fulltext=Identification+of+TonB+Homologs&and

orexactfulltext=and&searchsubmit=redo&resourcetype=1&search=Search&author1=Larsen&

pubdate_year=1996&volume=&firstpage=&src=ml

24. 我能使用ChIP试剂盒做组织样品么？

答：可以。ChIP试剂盒已被成功用于肝脏、脾脏、结肠和全鼠胚胎。

25. Q: EZ ChIP试剂盒和常规ChIP试剂盒之间的区别是什么？

答： 关键区别在于DNA的纯化方式，EZChIP用纯化柱纯化DNA。

26. 进行IgG纯化时蛋白质A和蛋白质G的区别是？

答： 蛋白质A结合到IgG的Fc部分。蛋白质G选择性结合到IgG的Fc部分，但也能结合到Fab区域，因此可用于纯化IgG1的Fab片段。 三、 实例讲解

下面以一篇文献中的几个结果为例，简要介绍一下ChIP的应用。

标题：

Estrogen Receptor-α DirectsOrdered, Cyclical,

and Combinatorial Recruitment of Cofactors on a NaturalTarget Promoter

来源：Cell

Figure 1 中A图说明的是在MCF-7（能表达hERα的人乳腺癌细胞），MDA-MB231（不能表达hERα的细胞）和MDA:: hERα（在MDA-MB231细胞中重新表达hERα）三种细胞系分别用EtOH（乙醇）和10 nM E2（estradiol，雌二醇）处理后，用real-time PCR检测pS2 mRNA的表达量，使用GAPDH做内参。结果显示在MCF-7细胞中用E2处理后pS2 mRNA表达量增加了12倍，而MDA-MB231细胞处理后未检测到pS2的表达，在MDA-MB231细胞中重新表达hERα（MDA:: hERα细胞）并处理后发现pS2能表达。

Figure 1中图B中Inputs即为细胞裂解液，作为PCR阳性对照（本次讲堂中步骤3.3），ChIP检测到在MCF7细胞和MDA:: hERα细胞表达pS2基因的同时，hERα蛋白和激活磷酸化 RNA 聚合酶 II（P-Pol II）能够与pS2基因的启动子序列结合在一起，acetylated histones（AC-Hist）是转录激活区域的标记分子。这个实验说明hERα是调控pS2基因的关键转录因子。

Figure 2 A讲解的是通过Re-ChIP筛选能够与pS2启动子序列结合的因子。第一次ChIP中所用的抗体标记在左边一排，Re-chIP中使用的抗体标记在图的上方。Re-ChIP就是把ChIP中IP下来的染色质蛋白抗体复合物使用强还原剂如DTT使复合物间的结合破坏，染色质重新游离，再次使用其他蛋白抗体进行ChIP实验。

关于ChIP和Re-ChIP的具体实验步骤，大家也可以参阅这篇文献，实验方法描叙得很详细。本次的讲座到此结束，由于本人能力和精力有限，本次讲堂中难免有不足的地方，欢迎大家修正补充。

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