1、ChIP常见问题汇总ChIP常见问题汇总 1 ChIP是什么答：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。它利用抗原抗体反应的特异性，可以真实地反映体内蛋白因子与基因组DNA结合的状况。2 ChIP有哪些应用答：近年来由于该技术不断的发展和完善，其应用范围已经从研究目的蛋白与已知靶序列间的相互作用，发展到研究目的蛋白与整个基因组的未知序列的相互作用；从研究一个目的蛋白与DNA的相互作用，发展到研究两个蛋白与DNA共同结合的相互作用；从研究启动子区域的组蛋白的修饰，发展到研究结合在DNA序列上的蛋白复
2、合物。3 ChIP技术的原理答：生理状态下把细胞内的DNA与蛋白质交联在一起，通过超声或酶处理将染色质切为小片段后，利用抗原抗体的特异性识别反应，将与目的蛋白相结合的DNA片段沉淀下来。染色质免疫沉淀技术一般包括细胞固定，染色质断裂，染色质免疫沉淀，交联反应的逆转，DNA的纯化，以及DNA的鉴定。因为ChIP实验涉及的步骤多，结果的重复性较低，所以对ChIP实验过程的每一步都应设计相应的对照，而且对结果的分析也需要有一定的经验。4 做ChIP试验，必须做甲醛固定么答：不一定，视样品及试验方案而定。做甲醛固定的为X-ChIP，而不需要固定的为N-ChIP。甲醛能有效的使蛋白质-蛋白质，蛋白质-D
3、NA，蛋白质-RNA交联，形成生物复合体，防止细胞内组分的重新分布。甲醛的交联反应是完全可逆的，便于在后续步骤中对DNA和蛋白质进行分析。甲醛的交联反应可被加入的甘氨酸终止。5 为什么必须将DNA切碎至少于1000bp大小（大约3个核小体 400-500bp）答：为确保ChIP实验有良好精度。若您的平均片段长度大于1000bp，您将会分离获得包含您目标序列的DNA，但所要研究的蛋白会离您目标序列有700个核苷酸的距离。6 为什么使用鲑鱼精子DNA来封闭琼脂糖珠子为什么我的样品中鲑鱼精子DNA不会发生PCR反应答：鲑鱼精子用于降低降低染色质DNA与琼脂糖珠子的非特异性结合。实验者不太可能对鲑鱼组
4、织做ChIP实验，所以此DNA不会因交叉杂交而被PCR引物扩增。7 引物最佳设计是什么样的答：引物长度应为24个核苷酸，应含有50%GC碱基对，Tm值为60C。不要扩增大于600-800个核苷酸的序列。不必考虑基因组内不独一序列。8 您推荐下如何从琼脂糖（或琼脂糖凝胶）中洗脱抗体-蛋白-DNA复合物，用来做re-ChIP试验答：在ChIP分析试剂盒内可找到洗脱缓冲液，用它洗脱复合物。对于re-ChIP，有必要添加蛋白酶抑制剂到免疫沉淀洗液和洗脱缓冲液，及第二轮实验用的稀释缓冲液。请确定所有溶液处于低温，蛋白质不会因此而在收集第一次免疫沉淀的复合物或第二次免疫沉淀时降解。9. 蛋白A琼脂糖能被用
5、于小鼠IgM答: 蛋白质A不能与小鼠IgM结合。可以考虑用一个桥接抗体连接。10. 在做ChIP之前，有办法纯化细胞核么答: 在细胞与甲醛交联后，细胞核可通过“溶胀缓冲液”培育及剪刀细胞均质器（dounce homogenization）制备（至少10倍体积）。溶胀缓冲液：25M Hepes, pH MgCl210mM KCl% NP-401mM DTT PMSF蛋白酶抑制剂混合剂然后按照protocol在SDS裂解液中裂解11: ChIP超声波的最佳条件答: 1. 确定裂解物在冰上放置了至少10分钟。不要震荡或者摇晃裂解物，避免有气饱（气泡产生）。超声波仪会替你做这些。2脉冲应在10秒（与体
6、积一致）。3. 样品量小于400 uL间隔应大于1分钟，在EP管中的更大体积间隔大于3分钟。4. 避免泡沫。请确定超声探头靠近液体底部（不能让探头碰到管壁）。5若发现泡沫，立即停止超声，置于冰上。旋转EP管以去除泡沫，继续超声。6. 在按“开始”键之前将探头置于液体中。12: 客户能只用哺乳动物细胞的细胞核而不是整个细胞么答: 是，实际上比起全细胞，细胞核更好。我们用全细胞裂解物，因为它更简便，通常也能得到好结果。此页有一些相关信息： 我是否可以改变逆转交联的时间和温度答：并不推荐少于4个小时的逆转交联. 但是, 可以将样本在65度过夜逆转交联.需要注意的是,样品不能干掉。14. 做组蛋白Ch
7、IP时, 什么时候不需要交联答：native ChIP中, Histone H3 and Histone H4 都不需要交联反应, 因为它们本身来说和DNA结合的非常紧密. 组蛋白H2A和H2B并不是紧密联接,但是在native ChIP中依然可以不需要交联反应.请ChIP初学者尤其注意此条！15. 什么是input DNA Output DNA答：从染色质上获得的未做过IP的已经被逆转交联的DNA. 它是检查PCR是否有效的对照。Output DNA是来自每次IP实验的DNA.其实就是genomic DNA. 在ChIP实验中, sonication 或酶解后, 样本取部分不做IP, 直接逆
8、转交联. 它的最主要的作用就是检查PCR系统是否work. 通常情况下, 在该条道中,都可以看到条带的.如果没有,说明PCR系统不work.16. 通过改善交联是否能提高ChIP的enrichment答：Not likely.Formaldehyde is a very reactive dipolar compound in which the carbon atom acts as a nucleophilic center for amino and imino groups of amino acids (Lys, Arg, and His) and of DNA (primarily
9、 A and C), leading to the formation of a Schiff base. This intermediate can further react with a second amino group, resulting in the final DNAprotein complex 1-3. Your protein, or the protein-DNA complex, also crosslink with other proteins and lipids, via the same mechanism,. Increasing formaldehyd
10、e concentration and/or the incubation time may adversely affect immunoprecipitation. It is recommended that you optimize other parts of the protocol for improvement.17. 如何来量化经IP后的DNA答：DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria
11、. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of (+/- . You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment t
12、o optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin. Also, do not use more than 20 cycles, making sure that dNTPs always remain in excess. Also, include each reaction a contr
13、ol primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and inc
14、lude no antibody control PCRs as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molec
15、ular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH for 30 minutes-no destaining is required. Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This
16、has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioa
17、ctive quantitation method is described in Suka et al (2001) Molec. Cell, 8: 473-479.18. 为什么ChIP试验需要用经验证的抗体答：抗原抗体之间的结合是通过抗体特异性的识别抗原表位并结合的，有的抗体识别的抗原表位比较小，不容易暴露，在ChIP实验中容易被DNA包裹，从而使抗体无法结合，因此用来做ChIP的抗体一般是需要经过chip实验验证的，商业化的抗体都应该是验证过的。19. 如何确保在最大功率下超声裂解不起泡1. Use the volume of SDS lysis buffer you choose,
18、cool on ice2. Start sonication with increasing power until foaming occurs.3. Lower the power a little. This is the power you can use (for instance, if it starts foaming at 40%, then 35% should be OK).4. Cool the above vial and sonicate for one pulse. Touch the vial without glove and it should not be
19、 hot (warm is OK), otherwise shorten the time (10-15 seconds are generally used).5. Prepare cells in lysis buffer and sonicate for 3, 6, 9 (or whatever you prefer) pulses and check DNA on gel.Other things to watch out:Load 1 x 105 cell equivalent (This is ug DNA)/Lane.Make sure you digested all the
20、RNA (The big smiley band)Load non-sonicated DNA in one lane.20. 什么是reChIP技术ChIP reChIP是在第一次ChIP的基础上不解交联，而继续进行另一个目的蛋白的免疫沉淀，从而得到与两种目的蛋白都结合的DNA序列。值得注意的是，因为 通过两次免疫沉淀富集的DNA量比较少，所以在分析时通常要把多次免疫沉淀的DNA浓缩后再进行操作。21. Protein L与Protein A, Protein G的区别答：Protein L is an immunoglobulin-binding protein that originat
21、es from the bacteria Peptostreptococcus magnus. Unlike Protein A and Protein G, which bind primarily through Fc regions ., heavy chain) of immunoglobilins, Protein L binds Igs through interactions with the light chains. Since no part of the heavy chain is involved in the binding interaction, Protein
22、 L binds a wider range of Ig classes than Protein A or G. Protein L binds to representatives of all classes of Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L. From this wide-ranging binding capability with respect to Ig class
23、es, Protein L is not a universal immunoglobilin-binding protein. Binding of Protein L to immunoglobulins is restricted to those containing kappa light chains ., k chain of the VL domain).1 In humans and mice, kappa (k) light chains predominate. The remaining immunoglobulins have lambda (l) light cha
24、ins. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For example, it binds human VkI, VkIII and VkIV subtypes but does not bind the VkII subtype. Binding of mouse immunoglobulins is restricted to those having VkI light chains.22. 关于酶切DNA片段，Micrococcal Nucl
25、ease Enzyme是什么It preferentially digests single-stranded DNA and AT or AU-rich regions but is also active against RNA and double-stranded DNA (all sequences are ultimately cleavable). Products of digestion are nucleic acid fragments containing 3 phosphate termini. Exhaustive digestion with excess enz
26、yme yields mono- and dinucleotides. The enzyme requires calcium ions for activity and is easily inactivated by EDTA or EGTA.Application. 1. Removal of nucleic acids, especially single-stranded DNA or RNA. 2. Preparation of mRNA-free protein synthesis system from rabbit reticulocyte lysates. 3. Study
27、 of chromatin structure.23 ChIP试剂盒适用于细菌么细菌应该也能用。之前有两篇论文可以做参考：Molecular basis for the exploitation of spore formation as survival mechanism by virulentphage phi29.WJ Meijer, V Castilla-Llorente, L Villar, H Murray, J Errington, and M SalasEMBO J, October 19, 2005; 24(20): 3647-57.&HITS=&hits=&RESULTF
28、ORMAT=1&author1=Meijer&andorexacttitle=and&fulltext=molecular+basis+for+the+exploitation+of+spore+formation+&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&resourcetype=HWCITIdentification of TonB homologs in the family Enterobacteriaceae and evidence forconservation of TonB-depen
29、dent energy transduction complexesRA Larsen, PS Myers, JT Skare, CL Seachord, RP Darveau, and K PostleJ. Bacteriol., Mar 1996; 178: 1363 - 1373.&andorexactfulltext=and&searchsubmit=redo&resourcetype=1&search=Search&author1=Larsen&pubdate_year=1996&volume=&firstpage=&src=ml24 我能使用ChIP试剂盒做组织样品么可以。ChIP
30、试剂盒已被成功用于肝脏、脾脏、结肠和全鼠胚胎。25. millipore的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，EZ ChIP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。27. 从细胞/组织中能够获得的多少genomic DNA通常情况下，单个的人细胞中可得到 DNA，百万细胞可得到7 ug. 4 mg肝组织相当于 x 106 liver cells (K562). 有经验的用户往
31、往可以从3 x 106 个细胞or 11 mg 肝组织中获取15-30 mg DNA (使用RNase A). 因为我们通常在ChIP实验中使用约20 ug染色质，您可以用2-5百万个细胞。如果您使用染色质来标准化ChIP实验，您就只需要最少的量. 如果您的PCR系统正常，就不会出现问题，因为ChIP只需少量染色质，实验标准化更加重要.28. 请问有没有人在转染导入真核细胞的质粒上做类似于ChiP的试验完全可以做，只要保证转染效率。但是转染的质粒在体内的行为和染色体基因是不一样的，因此用这种方法可能得到不同的结果。29. 您推荐下如何从琼脂糖（或琼脂糖凝胶）中洗脱抗体-蛋白-DNA复合物，用来
32、做re-ChIP试验答: 在ChIP分析试剂盒内可找到洗脱缓冲液，用它洗脱复合物。对于re-ChIP，有必要添加蛋白酶抑制剂到免疫沉淀洗液和洗脱缓冲液，及第二轮实验用的稀释缓冲液。请确定所有溶液处于低温，蛋白质不会因此而在收集第一次免疫沉淀的复合物或第二次免疫沉淀时降解。30. 您为何（为什么）使用鲑鱼精子DNA来封闭琼脂糖珠子为什么我的样品中鲑鱼精子DNA不会发生PCR反应答: 鲑鱼精子用于降低降低染色质DNA与琼脂糖珠子的非特异性结合。实验者不太可能对鲑鱼组织做ChIP实验，所以此DNA不会因交叉杂交而被PCR引物扩增。31.密理博EZ-ChIP试剂盒中抗组蛋白H3抗体也可用于大鼠么答: 是的，该抗体100%在大鼠中100%保守。32.密理博EZ ChIP试剂盒和常规ChIP试剂盒之间的主要区别是什么答: 关键区别在于DNA的纯化方式，EZ ChIP用纯化柱纯化DNA。
