ChIP常見問題匯總

1、ChIP常見問題匯總 1    ChIP是什么？答：染色質(zhì)免疫沉淀技術(shù)（Chromatin Immunoprecipitation，簡稱ChIP）是研究體內(nèi)蛋白質(zhì)與DNA相互作用的一種技術(shù)。它利用抗原抗體反應(yīng)的特異性，可以真實地反映體內(nèi)蛋白因子與基因組DNA結(jié)合的狀況。2    ChIP有哪些應(yīng)用？答：近年來由于該技術(shù)不斷的發(fā)展和完善，其應(yīng)用范圍已經(jīng)從研究目的蛋白與已知靶序列間的相互作用，發(fā)展到研究目的蛋白與整個基因組的未知序列的相互作用；從研究一個目的蛋白與DNA的相互作用，發(fā)展到研究兩個蛋白與DNA共同結(jié)合的相互作用；從研究啟動子區(qū)域的組蛋白的修飾，

2、發(fā)展到研究結(jié)合在DNA序列上的蛋白復(fù)合物。3    ChIP技術(shù)的原理？答：生理狀態(tài)下把細(xì)胞內(nèi)的DNA與蛋白質(zhì)交聯(lián)在一起，通過超聲或酶處理將染色質(zhì)切為小片段后，利用抗原抗體的特異性識別反應(yīng)，將與目的蛋白相結(jié)合的DNA片段沉淀下來。染色質(zhì)免疫沉淀技術(shù)一般包括細(xì)胞固定，染色質(zhì)斷裂，染色質(zhì)免疫沉淀，交聯(lián)反應(yīng)的逆轉(zhuǎn)，DNA的純化，以及DNA的鑒定。因為ChIP實驗涉及的步驟多，結(jié)果的重復(fù)性較低，所以對ChIP實驗過程的每一步都應(yīng)設(shè)計相應(yīng)的對照，而且對結(jié)果的分析也需要有一定的經(jīng)驗。4    做ChIP試驗，必須做甲醛固定么？答：不一定，視樣品及試驗方案而定。做甲醛

3、固定的為X-ChIP，而不需要固定的為N-ChIP。甲醛能有效的使蛋白質(zhì)-蛋白質(zhì)，蛋白質(zhì)-DNA，蛋白質(zhì)-RNA交聯(lián)，形成生物復(fù)合體，防止細(xì)胞內(nèi)組分的重新分布。甲醛的交聯(lián)反應(yīng)是完全可逆的，便于在后續(xù)步驟中對DNA和蛋白質(zhì)進(jìn)行分析。甲醛的交聯(lián)反應(yīng)可被加入的甘氨酸終止。5    為什么必須將DNA切碎至少于1000bp大?。ù蠹s3個核小體 400-500bp）?答：為確保ChIP實驗有良好精度。若您的平均片段長度大于1000bp，您將會分離獲得包含您目標(biāo)序列的DNA，但所要研究的蛋白會離您目標(biāo)序列有700個核苷酸的距離。6    為什么使用鮭魚精子DNA來封

4、閉瓊脂糖珠子？為什么我的樣品中鮭魚精子DNA不會發(fā)生PCR反應(yīng)？答：鮭魚精子用于降低降低染色質(zhì)DNA與瓊脂糖珠子的非特異性結(jié)合。實驗者不太可能對鮭魚組織做ChIP實驗，所以此DNA不會因交叉雜交而被PCR引物擴增。7    引物最佳設(shè)計是什么樣的？答：引物長度應(yīng)為24個核苷酸，應(yīng)含有50%GC堿基對，Tm值為60°C。不要擴增大于600-800個核苷酸的序列。不必考慮基因組內(nèi)不獨一序列。8    您推薦下如何從瓊脂糖（或瓊脂糖凝膠）中洗脫抗體-蛋白-DNA復(fù)合物，用來做re-ChIP試驗？答：在ChIP分析試劑盒內(nèi)可找到洗脫緩沖液，用它洗脫復(fù)合

5、物。對于re-ChIP，有必要添加蛋白酶抑制劑到免疫沉淀洗液和洗脫緩沖液，及第二輪實驗用的稀釋緩沖液。請確定所有溶液處于低溫，蛋白質(zhì)不會因此而在收集第一次免疫沉淀的復(fù)合物或第二次免疫沉淀時降解。9. 蛋白A瓊脂糖能被用于小鼠IgM?答: 蛋白質(zhì)A不能與小鼠IgM結(jié)合?？梢钥紤]用一個橋接抗體連接。10. 在做ChIP之前，有辦法純化細(xì)胞核么？答: 在細(xì)胞與甲醛交聯(lián)后，細(xì)胞核可通過“溶脹緩沖液”培育及剪刀細(xì)胞均質(zhì)器（dounce homogenization）制備（至少10倍體積）。溶脹緩沖液：25M Hepes, pH 7.81.5mM MgCl210mM KCl0.1% NP-401mM DT

6、T0.5mM PMSF蛋白酶抑制劑混合劑然后按照protocol在SDS裂解液中裂解11: ChIP超聲波的最佳條件？答: 1. 確定裂解物在冰上放置了至少10分鐘。不要震蕩或者搖晃裂解物，避免有氣飽（氣泡產(chǎn)生）。超聲波儀會替你做這些。2脈沖應(yīng)在10秒（與體積一致）。3. 樣品量小于400 uL間隔應(yīng)大于1分鐘，在EP管中的更大體積間隔大于3分鐘。4. 避免泡沫。請確定超聲探頭靠近液體底部（不能讓探頭碰到管壁）。5若發(fā)現(xiàn)泡沫，立即停止超聲，置于冰上。旋轉(zhuǎn)EP管以去除泡沫，繼續(xù)超聲。6. 在按“開始”鍵之前將探頭置于液體中。12: 客戶能只用哺乳動物細(xì)胞的細(xì)胞核而不是整個細(xì)胞么？答: 是，實際上

7、比起全細(xì)胞，細(xì)胞核更好。我們用全細(xì)胞裂解物，因為它更簡便，通常也能得到好結(jié)果。此頁有一些相關(guān)信息：13. 我是否可以改變逆轉(zhuǎn)交聯(lián)的時間和溫度?答：并不推薦少于4個小時的逆轉(zhuǎn)交聯(lián). 但是, 可以將樣本在65度過夜逆轉(zhuǎn)交聯(lián).需要注意的是,樣品不能干掉。14. 做組蛋白ChIP時, 什么時候不需要交聯(lián)?答：native ChIP中, Histone H3 and Histone H4 都不需要交聯(lián)反應(yīng), 因為它們本身來說和DNA結(jié)合的非常緊密. 組蛋白H2A和H2B并不是緊密聯(lián)接,但是在native ChIP中依然可以不需要交聯(lián)反應(yīng).請ChIP初學(xué)者尤其注意此條！15. 什么是input DNA?

8、Output DNA?答：從染色質(zhì)上獲得的未做過IP的已經(jīng)被逆轉(zhuǎn)交聯(lián)的DNA. 它是檢查PCR是否有效的對照。Output DNA是來自每次IP實驗的DNA.其實就是genomic DNA. 在ChIP實驗中, sonication 或酶解后, 樣本取部分不做IP, 直接逆轉(zhuǎn)交聯(lián). 它的最主要的作用就是檢查PCR系統(tǒng)是否work. 通常情況下, 在該條道中,都可以看到條帶的.如果沒有,說明PCR系統(tǒng)不work.16. 通過改善交聯(lián)是否能提高ChIP的enrichment?答：Not likely. Formaldehyde is a very reactive dipolar com

9、pound 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 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 compl

10、ex 1-3. Your protein, or the protein-DNA complex, also crosslink with other proteins and lipids, via the same mechanism,. Increasing formaldehyde concentration and/or the incubation time may adversely affect immunoprecipitation. It is recommended that you optimize other parts of the protocol for imp

11、rovement.17. 如何來量化經(jīng)IP后的DNA?答：DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear

12、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 to 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

13、 of antibody and input chromatin. Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs

14、 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 include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where y

15、ou 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 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required. Quantitation i

16、s 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 has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained ge

17、ls 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 radioactive quantitation method is described in Suka et al (2001) Molec. Cell, 8: 473-479.18. 為什么ChIP試驗需要用經(jīng)驗證的抗體？答：抗原抗體之間的結(jié)合是通過抗體特異性的識別抗

18、原表位并結(jié)合的，有的抗體識別的抗原表位比較小，不容易暴露，在ChIP實驗中容易被DNA包裹，從而使抗體無法結(jié)合，因此用來做ChIP的抗體一般是需要經(jīng)過chip實驗驗證的，商業(yè)化的抗體都應(yīng)該是驗證過的。19. 如何確保在最大功率下超聲裂解不起泡1. Use the volume of SDS lysis buffer you choose, cool on ice 2. Start sonication with increasing power until foaming occurs. 3. Lower the power a little. This is the po

19、wer 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 hot (warm is OK), otherwise shorten the time (10-15 seconds are generally used). 5. Prepare cells in lysi

20、s 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 <0.7 ug DNA)/Lane. Make sure you digested all the RNA (The big smiley band) Load non-sonicated DNA in one lane.20. 什么是reChIP

21、技術(shù)？ChIP reChIP是在第一次ChIP的基礎(chǔ)上不解交聯(lián)，而繼續(xù)進(jìn)行另一個目的蛋白的免疫沉淀，從而得到與兩種目的蛋白都結(jié)合的DNA序列。值得注意的是，因為 通過兩次免疫沉淀富集的DNA量比較少，所以在分析時通常要把多次免疫沉淀的DNA濃縮后再進(jìn)行操作。21. Protein L與Protein A, Protein G的區(qū)別答：Protein L is an immunoglobulin-binding protein that originates from the bacteria Peptostreptococcus magnus. Unlike Protein A and Pro

22、tein G, which bind primarily through Fc regions (i.e., 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 L binds a wider range of Ig classes than Protein A or G. Protein L

23、binds to representatives of all classes of Ig, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFvDespite this wide-ranging binding capability with respect to Ig classes, Protein L is not a universal immunoglobilin-binding protein. Binding of Protein L to immunoglobulins is r

24、estricted to those containing kappa light 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) light chains. Furthermore, Protein L is effective in binding only certain subtypes of kappa light chains. For exam

25、ple, 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. 關(guān)于酶切DNA片段，Micrococcal Nuclease Enzyme是什么？It preferentially digests single-stranded DNA and AT or AU-rich regions but is also active

26、 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 enzyme yields mono- and dinucleotides. The enzyme requires calcium ions for activity and is easily ina

27、ctivated 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 of chromatin structure.23 ChIP試劑盒適用于細(xì)菌么？細(xì)菌應(yīng)該也能用。之前有兩篇論文可以做參考：Molecular basis for the exploit

28、ation 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./cgi/medline/pmid;16193065?maxtoshow=&HITS=&hits=&RESULTFORMAT=1&auth

29、or1=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 forconservati

30、on of TonB-dependent energy transduction complexesRA Larsen, PS Myers, JT Skare, CL Seachord, RP Darveau, and K PostleJ. Bacteriol., Mar 1996; 178: 1363 - 1373./cgi/searchresults?fulltext=Identification+of+TonB+Homologs&andorexactfulltext=and&searchsubmit=redo&resourcetype=1&search=Search&author1=Larsen&pubdate_year=1996&volume=&firstpage=&src=ml24 我能使用ChIP試劑盒做組織樣品么？可以