表觀2016chip實(shí)驗(yàn)指南

1、技術(shù)指南染色質(zhì)免疫沉淀(ChIP)指南：成功的關(guān)鍵關(guān)注 密理博 查抗體技術(shù)及現(xiàn)貨信息盡掌握Calbiochem 小分子 大智慧Chemicon品質(zhì) 成就發(fā)現(xiàn)UpS e 歷史悠久 精益求精信號(hào)通路抑制劑行家之選干細(xì)胞和神經(jīng)研究金標(biāo)準(zhǔn)表觀遺傳及信號(hào)通路研究領(lǐng)跑者穩(wěn)定 放心 創(chuàng)新目錄2簡(jiǎn)介3染色質(zhì)免疫沉淀4ChIP的靶點(diǎn)5ChIP中的組蛋白5ChIP實(shí)驗(yàn)83.1. 樣品10收集樣本10蛋白質(zhì)與DNA交聯(lián)11細(xì)胞裂解11染色質(zhì)斷裂12免疫沉淀13單克隆或多克隆抗體?13ChIP的對(duì)照抗體14特異性檢測(cè)14驗(yàn)證檢測(cè)16ChIP抗體的應(yīng)用17洗滌免疫沉淀物18洗脫和交聯(lián)逆轉(zhuǎn)18實(shí)時(shí)定量PCR (qPCR)

2、19qPCR反應(yīng)的效率20數(shù)據(jù)分析21Troubleshooting22補(bǔ)充操作25ChIP實(shí)驗(yàn)的細(xì)胞25ChIP實(shí)驗(yàn)的組織25優(yōu)化超聲處理和分析DN段26DNA純化26簡(jiǎn)化ChIP的試劑盒及分析27一天完成的ChIP試劑盒27用于組織的ChIP試劑盒27高通量ChIP試劑盒28全組ChIP試劑盒 二代29全組ChIP試劑盒 分析29Absurance 組蛋白H2、H3、H4抗體特異性30表觀遺傳學(xué)研究新技術(shù)及工具311關(guān)的 的的功關(guān)的僞的成成染色質(zhì)染色質(zhì)成的 147 bp DNAH2AH2BH3H4 的成染色質(zhì)的儎的染色質(zhì)DNA的染色質(zhì) 質(zhì)DNA染色質(zhì)的DNA因的的的 偵的亾匔匔DNA的DN

3、A偵染色質(zhì) RNA的因的因 的的的因成 因亱的人的Prader-Willi功3Upse(唎的90ChIP唎ChIP技術(shù)成：儎 的僂 成僂 儎ChIP ChIP僂的因 A, GA/G的ChIP 的RettFragile X唎ICFAngelmanBeckwith-Wiedmann伕匔亾1.1 染色質(zhì)免疫沉淀的染色質(zhì)免疫沉淀ChIP的ChIP 的質(zhì)-DNA質(zhì)的ChIP質(zhì)DNA的的免疫沉淀 qPCR ChIP-chipChIP-seq 的DNA僂的ChIP亽僚的技術(shù)儎質(zhì)的染色質(zhì)的的的的的僂僂指ChIP的從DNAChIP A.B.450400350300250200150100500605040302

4、0100389.854731292827181211IgGHP11. (A) 染色質(zhì)1d(HP1)的因關(guān)的DNA的成功ChIP唎的ChIPAb+ Hp1/啖IgG唎的Magna ChIP G kit 17-10085的(B) 質(zhì)的儎96ChIP的因唎的panelMagna ChIP HT96 Kit17-100774Feonltd EnriFeonltd EnriControl IgGE2F3 (HeLa)pCREB (HeLa)LSF (Mouse Brain)H3K9Ac (HeLa)FoxA2 (Mouse Liver)H3T11P (Treated HeLa)JMJD1C (HeLa)

5、CHIP ChIP：的DNA指的質(zhì)因因的因關(guān)的的DNA/質(zhì) 質(zhì)的偵染色質(zhì)因染色的ChIP的22.1 ChIP的偵的的ChIP因功關(guān)的偵 ：H1H2H3H4 H234成的H1染色質(zhì)的偵PTM：素SUMOChIP儎的染色質(zhì)的的質(zhì)的偵因質(zhì) ChIP因質(zhì)關(guān)ChIP的的關(guān)鍵的ChIP偵因因DNA的質(zhì)素2關(guān)com/antibodies偵PTM染色質(zhì)的 的HAT DNA的染色質(zhì) 因HDAC H2AH2BH3H4的偵的ChIP僂ChIP的質(zhì)從：的ChIP 偵的 儎的 DNA質(zhì) 的 染色質(zhì)關(guān)的2. 關(guān)的偵Lam et al, (2007).com/nsmb/journal/v14/n11/f ull/nsmb1

6、307.htmlLysine (K)Arginine (K)Glutamic Acid (E)Proline (P)Serine (S)Threonine (T)CitrullinationAcetylation SUMOylation Ubiquitination Biotinylation Crotonylationcis-trans isomerizationos orylationMethylationRiylation5的因 的H3927的染色質(zhì)成的質(zhì)b group的關(guān)H3436的染色因trithorax的關(guān)acacacub1N G R GQ G CA R A H2A K T E S

7、 H H K A K G KCacacacacub1NP E P A K SK G S K KK A H2B K A V T K Y T S S KC151214 1520120acme meme acmeme ac acK56memeH3的染色質(zhì)DNA 的H1H41的H31028的儎染色質(zhì)的 染色質(zhì)的 H3的10：G2 的 DNAH2A.X139的ATM/ATKDNA的DNA的 偵 的的偵偵的的偵的關(guān) “histone code” 染色質(zhì)DNA的NA R T KR K S TKR KKR K SK2 3 48 9 10 11 14 17 18 23 26 27 28 36K79meacaca

8、cacmeH4NS G R G K G G K G L G K G G A K R H R K VC135812161820 3: 的偵的：acub 素meLam et al. , 2007 htt/nsmb/journal/v14/n11/full/nsmb1307.html1501211005010IgGH3S10P4. 唎的ChIPAb+os o-Histone H3 (Ser10) /17-685啖IgG唎的 Magna ChIP A/G Kit 17-10085的6Feonltd EnriChIP的成功ChIPAb+ChIP僂的/DescriptionCatalogue No.ChI

9、PAb+ Histone H2A.Z17-10048ChIPAb+ Histone H2B17-10054ChIPAb+ Histone H3 (C-term)17-10046ChIPAb+ Histone H3 (Unmod Lys4)17-675ChIPAb+ Acetyl Histone H317-615ChIPAb+ Acetyl-Histone H3 (Lys4)17-10050ChIPAb+ Acetyl-Histone H3 (Lys9)17-658ChIPAb+ Acetyl-Histone H3 (Lys14)17-10051ChIPAb+ Monomethyl Histon

10、e H3 (Lys27)17-643ChIPAb+ Dimethyl-Histone H3 (Lys4)17-677ChIPAb+ Dimethyl-Histone H3 (Lys9)17-648ChIPAb+ Trimethyl-Histone H3 (Lys4)17-614ChIPAb+ Trimethyl-Histone H3 (Lys4)17-678ChIPAb+ Trimethyl-Histone H3 (Lys9)17-625ChIPAb+ Trimethyl-Histone H3 (Lys27)17-622ChIPAb+ Trimethyl-Histone H3 (Lys36)1

11、7-10032ChIPAb+ Trimethyl-Histone H3 (Lys79)17-10130ChIPAb+ os o-Histone H3 (Ser10)17-685ChIPAb+ Acetyl Histone H417-630ChIPAb+ Acetyl-Histone H4 (Lys5)17-10045ChIPAb+ 僂的 免疫僂的200com/genetics關(guān) 的ChIPAb+ ：DescriptionCatalogue No.ChIPAb+ Acetyl-Histone H4 (Lys5)17-10045ChIPAb+ Acetyl-Histone H3 (Lys14),1

12、7-10051ChIPAb+ Acetyl-Histone H4 (Lys8)17-10099ChIPAb+ Acetyl-Histone H4 (Lys16)17-10101ChIPAb+ Dimethyl-Histone H3 (Lys27),17-10108ChIPAb+ Sp117-601ChIPAb+ ERa17-603ChIPAb+ HDAC117-608ChIPAb+ Trimethyl-Histone H3 (Lys4)17-614ChIPAb+ Acetyl Histone H317-615ChIPAb+ RNA Polymerase II17-620ChIPAb+ Trim

13、ethyl-Histone H3 (Lys27)17-622ChIPAb+ Trimethyl-Histone H3 (Lys9)17-625ChIPAb+ Acetyl Histone H417-630ChIPAb+ Dimethyl-Histone H3 (Lys9)17-648ChIPAb+ Acetyl-Histone H3 (Lys9)Puri ed17-658ChIPAb+ EZH2, clone AC2217-662ChIPAb+ Dimethyl-Histone H3 (Lys4)17-677ChIPAb+ Acetyl-Histone H3 (Lys27)17-6837ChI

14、P僂3ChIP僂5僚：染色質(zhì)免疫沉淀DNAPCR的ChIP僂5僚2-3 成1成 的ChIP僂的1的ChIP僂Magna ChIP protein A/G kit 17-10085EZ-Magna ChIP protein A/G kit 17-10086 的 PCR 的免疫沉淀的儎ChIP僂的-僂的關(guān)的Magna ChIP 17-10086關(guān)染色質(zhì)免疫沉淀的:com/genetics8僚 1 2-30.50.5 染色質(zhì)0.760.76 免疫沉淀2 3 3 DNAPCR3 3 的ChIP僂：的的Protein A/Protein G的K的PCR的染色質(zhì)免疫沉淀：ChIPChIP僂的唎 ChIPC

15、hIP的僂的俌因的Magna ChIP質(zhì)的ChIP的：僂troubleshooting 僂的ChIP僂1成儎的96ChIP成：EZ-Magna ChIPEZ-ChIPIgG 的qPCR 因ChIP-seq 的技術(shù)troubleshootingDescriptionCatalogue No.Magna ChIP A Kit17-610Magna ChIP G Kit17-611EZ-Magna ChIP A Kit17-408EZ-Magna ChIP G Kit17-409Magna ChIP A/G Kit17-10085EZ-Magna ChIP A/G Kit17-10086Magna

16、ChIP HT96 Kit17-10077EZ-Magna ChIP HT96 Kit17-10078Magna ChIP-Seq Chromatin Immunoprecipiion and Next Generation Sequencing Library Preparation Kit17-1010Magna ChIP2 Universal Chromatin Immunoprecipiion DNA Microarray Kit17-1000Magna ChIP2 Universal Chromatin Immunoprecipiion DNA Microarray Quad Kit

17、17-1004Magna ChIP G Tie Kit17-20000Magna ChIP Protein A+G Magnetic Beads16-663Magna ChIP Protein G Magnetic Beads16-662Magna ChIP Protein A Magnetic Beads16-661ChIP Assay Kit (Agarose)17-295EZ-ChIP Kit (Agarose)17-371關(guān)ChIP的27-30享93.1. 3.1.1. ：的ChIP僂的/ChIP僂的亱的質(zhì)因 的的染色質(zhì)亱ChIP ChIP的質(zhì)的ChIP的染色質(zhì)的 IgG的儎質(zhì)的ChI

18、P勒成1-3mm3的ChIP的 的質(zhì)的 的ChIP的仍的107100 ChIP僂的的 的染的質(zhì)的 的的的的的質(zhì)儎質(zhì)的儎 的RNAII的偵ChIP1x104成-功的 12 的質(zhì)的指南1: ChIP1的指南400350300250200150100500GAPDHUpstr(-)CortexHeartTieKidney2: 的指南5：RNAIIGAPDH的Magna ChIP G Ti e Kit 17-20000 Anti-RNA Polymerase II clone CTD4H8 05-623B僂1 g免疫沉淀啖的染色質(zhì)啖GAPDH的qPCR免疫沉淀的DNAqPCR免疫沉淀的染色質(zhì)僂 IgG

19、關(guān)DNA的Dhfr因的UpStr (-)qPCR的的ChIP DNA的qPCR 105ChIP-seq1-10 ng106ChIP-chip10-100 ng1081. 儎質(zhì)儎的10Feonltd Enri質(zhì)的的/ChIP儎儎104偵的RNAII105-106因偵因 TFIIDPCG106-107的因107 因3.1.2. 質(zhì)DNA：質(zhì)1$的：質(zhì)DNA的 的質(zhì)的DNA 的質(zhì) DNA僂的質(zhì)/DNAnative ChIPN-ChIPN-ChIP偵的倉(cāng)的偵的DNA的質(zhì) ChIPX-ChIP的X-ChIP的 俌因 偵的ChIP 的DNA的2 0.2 nm的DNA的質(zhì)DNA的成質(zhì)/DNA的的僂的質(zhì)/DN

20、A 的3.1.3. ：/的質(zhì)/DNA的- 的dounce Mini-beadbeater- 的的的亽的質(zhì) 成的ChIP的成功的染色質(zhì)的ChIP指儎SDS的指的11CHIP的1% 1020的質(zhì) 質(zhì)質(zhì)DNA的的：的成色沉淀沉淀的質(zhì)的 的ChIP的僚的質(zhì)/DNA的的 vs. 的 的人3.1.4. 染色質(zhì)：染色質(zhì)成ChIP的200 1000 bp儎染色質(zhì)的質(zhì)/DNA的的ChIP的N-ChIP X-ChIP3.1.2N-ChIP 的DNA因的/DNAX-ChIP 的DNA 亱僂的染色質(zhì)的6：染色質(zhì)的2001000 bp127：2 1 沉淀 的DNA的 僂 DNA1000 bp200 bp12CHIP的亽

21、的的 的的的的的 功 vs. /200 1000 bp的 DNA的因100 200 bp的的成功僂功的因的的功儎功免疫沉淀僚的的ChIP因染色質(zhì) 免質(zhì)的染色質(zhì) 免功儎DNA的的倉(cāng)的僚DNA的的質(zhì):DNA:RNA的質(zhì):沉淀DNA染色質(zhì)染色質(zhì)的3.2. 免疫沉淀：的染色質(zhì)質(zhì)/DNA因的的唎免疫的從的的ChIP的 的的ChIP關(guān)鍵 的3.2.1. 的ChIPChIP僂成功的關(guān)鍵僚儎質(zhì)的Western blot僂 ChIPChIP僂的的的質(zhì) ChIP僂ChIPAb+僂的/的的ChIP僂的ChIP質(zhì)的的儎的 的因 質(zhì)的的的僚的質(zhì)的DNA的 17-10237 ChIPab+ HDAC2 Titration

22、 8： 的 僂的ChIP質(zhì)的因因 因的 亱的 免疫 關(guān)的從的 的的 質(zhì)的 的儎的免疫0.80.6560.2220.20.0130.006 0.0060.0FoldEnrient13Percent Input1 g lgG2 g lgG4 g lgG1 g HDAC22 g HDAC24 g HDAC2HDAC2 ChIP on CDKN1A (p21)promoter in U2OS cellsChIPChIP僂亱的的的質(zhì)的免疫沉淀成功儎的的的的8的ChIP質(zhì)的ChIP的僂因Western blot染色質(zhì)免疫沉淀Westernblot的質(zhì)ChIP的亱的質(zhì)3.2.2. Ch

23、IP的染色質(zhì)質(zhì)成的的ChIP僂染色質(zhì)的免疫沉淀ChIP僂的DNA 的ChIP的 ChIP僂：關(guān)的AbSurance 16-66516-66816-667PVDF89H2H3H4的儎質(zhì)Westernblot的的的關(guān) AbSurance的30享：的ChIP的nave的IgG的的啖ChIP的啖IgG的的ChIP的的IgG的IgG “”的的IgG的IgG的的910H3Lys4的的AbSurance的 H3Lys4的 H3Lys4的的1012345678(kDa) 260 160 110 80 ：的儎的質(zhì)RNAII H3H4 N僂ChIP的60 僂的50 40 30 20 15 3.2.3. 亱的偵的質(zhì)

24、偵的的亱的偵的10： H3 Lys45的H3Lys4的指 4的僂 僂偵的 的 的 1：2：偵的H3Lys43：H3Lys44：H3Lys45：H3Lys46：H3Lys97：H3Lys278：H4Lys20指/H3Lys417 kDa14Trimethyl Histone H3 (Lys4)123456789101112ABCDEFGHLayout of the AbSurance Histone H3 Antibody Speci city Array101112A100 ngB10 ngC100 ngD10 ngE100 ngF10 ngG100 ngH10 ng10：?jiǎn)o的AbSuranc

25、e H3 16-667H3Lys4色的 H3Lys4色的IgG 12H的色H3 1-19unmodH3 1-19R2me1H3 1-19R2me2aH3 1-19R2me2sH3 1-19 T3PH3 1-19K4acH3 1-19K4me1H3 1-19K4me2H3 1-19K4me3H3 1-19R8me1H3 1-19R8me2aH3 1-19R8me2sH3 1-19unmodH3 1-19R2me1H3 1-19R2me2aH3 1-19R2me2sH3 1-19 T3PH3 1-19K4acH3 1-19K4me1H3 1-19K4me2H3 1-19K4me3H3 1-19R8

26、me1H3 1-19R8me2aH3 1-19R8me2sH3 1-19K9acH3 1-19K9me1H3 1-19K9me2H3 1-19K9me3H3 1-19 S10PH3 1-19 T11PH3 7-26unmodH3 7-26K14acH3 7-26R17me1H3 7-26R17me2aH3 7-26R17me2sH3 7-26K18acH3 1-19K9acH3 1-19K9me1H3 1-19K9me2H3 1-19K9me3H3 1-19 S10PH3 1-19 T11PH3 7-26unmodH3 7-26K14acH3 7-26R17me1H3 7-26R17me2aH

27、3 7-26R17me2sH3 7-26K18acH3 16-34unmodH3 16-34K23acH3 16-34K27acH3 16-34K27me1H3 16-34K27me2H3 16-34K27me3H3 16-34R26me1H3 16-34R26me2aH3 16-34R26me2sH3 16-34 S28PH3 26-44unmodH3 26-44K36acH3 16-34unmodH3 16-34K23acH3 16-34K27acH3 16-34K27me1H3 16-34K27me2H3 16-34K27me3H3 16-34R26me1H3 16-34R26me2aH

28、3 16-34R26me2sH3 16-34 S28PH3 26-44unmodH3 26-44K36acH3 26-44K36me1H3 26-44K36me2H3 26-44K36me3H3 26-44 Y41PH3 47-65unmodH3 47-65K56acH3 71-89unmodH3 71-89K79me1H3 71-89K79me2H3 71-89K79me3Rat IgG 10 ngSheep IgG 10 ngH3 26-44K36me1H3 26-44K36me2H3 26-44K36me3H3 26-44 Y41PH3 47-65unmodH3 47-65K56acH3

29、 71-89unmodH3 71-89K79me1H3 71-89K79me2H3 71-89K79me3Mouse IgG 10 ngRabbit IgG 10 ng3.2.4. 僂ChIP的僂ChIP ChIPChIP的的因僂ChIP的免疫沉淀IP免疫IF免疫IHC的僂Western blot僂的的ChIP的僂 ChIP的成功因成功的ChIP亱 的ChIP的的ChIP的僂：Anti-JMJD6ChIPAb+ 17-1026312：Western blot Anti-JMJD6 JMJD6免疫沉淀 DNA的唎的Anti-JMJD609-812免疫WesternblotChIPAb+ JMJD

30、6/ 17-10263 Magna ChIP A Kit 17-408ChIPHeLa250221200150A4311005010IgGJMJD613：ChIP的JMJD6JMJD6DNA的11：免疫Anti-JMJD6的16Feonltd EnriChIP免疫Western blot/免疫沉淀成功的ChIP免疫的ChIP成功的ChIP的 的3.2.5. ChIP的Magnetic BeadsAgarose BeadsChIP A/G的的 ChIPChIP的 的染色質(zhì)質(zhì)/DNA MagnaGrIPRack 20-400PureProteome Magnetic Stand LSKMAGS08

31、LSKMAGS15MagnaGrIP HT96 Rack 17-10071DisadvantagesDisadvantagesSlightly higher costBlocking requiredPreclearing requiredMagnetic rack requiredNot visibleubes-porous (binding capacity dependent on bead surface area)High probability of bead loss during handlingA儎的G的 的啖IgG A/G的 因AG的 的A/GAG的的pull down的1

32、4的 的 人唎ChIP 7享的 的AGA G的A/G的僂的的A/G：17-646 ChIPAb+ Hp1gCBX3G的啖Protein A, G vs. A/GHeLa 1e6, 17-646 Hp1g GAPDH CodingProtein A, G vs. A/GHeLa 1e6, 17-646 Hp1g GAPDH Coding12004098236.69Similar ChIP signal, Lower background on bead blend Optimal at 20 uL blended beads35.4734.693532.933041625.8426.1125.7

33、625.44259111120AlgGAGGHp1g lgG Hp1g20A/G lgG2040A/GA/GHp1g lgG40A/G Hp1gAlgGAGHp1g lgGG20Hp1g A/G lgG2040A/GA/GHp1g lgG40A/G Hp1g14：AGA/G的ChIP的AGA/GIgGCq儎A/GAG的Cq的的A/G的儎17Mean CqFeonltd EnriAdvantagesHigh capacity binding (porous), whiay also increasespeci c bindingSimple equipment required (centrif

34、ugation or ltration)Slightly lower costAdvantagesLowspeci c bindingBlocking and preclearing not requiredEasy to handle during washesBeads are visibleubeReproducible results3.2.6. 免疫沉淀免疫沉淀AG的關(guān)因亱ChIP的僚的免疫沉淀 的染色質(zhì)質(zhì)因的儎的 ChIP的 儎的TE的的的 的：的 的 ChIP的ChIP的質(zhì)ChIP的質(zhì)的ChIP 的亰的ChIP4C染色質(zhì) 4C1 ChIP 的的ChIP染色質(zhì) 的A/GDescri

35、ptionCatalogue No.Magna ChIP Protein A+G Magnetic Beads16-663Magna ChIP Protein G Magnetic Beads16-662Magna ChIP Protein A Magnetic Beads16-661關(guān)ChIP的：om/genetics3.3. PureProteome Magnetic Stand LSKMAGS08儎的：的EP的 儎300 L的儎的：的的的 ：的1.5 mL2.0 mL僚的的染色質(zhì)ChIP的DNA染色質(zhì)的質(zhì)的成成/質(zhì)/DNA的的質(zhì)的質(zhì) 因成的成儎Magna GrIP Rack 20-400

36、儎的：4：15 mL0.5 mL：的81.5 - 2.0 mLqPCR亱DNA的K（Cat# 539480-100mg）的18DNA的: DNA DNAChelex的質(zhì)/DNAKRNAse僚 ChIP染的RNA的qPCRSYBR Green技術(shù) TaqMan 技術(shù)SYBR GreenDNA染DNA因DNA的 TaqMan 的 的PCR的的因的 DNA的53的的成的qPCR技術(shù) 的成3.4. PCRqPCR成ChIP的DNAPCRqPCR 的DNAqPCR的DNA TaqMan: SYBR Green染DNADNA 的成的因的的DNA qPCR 的成的 的DNA成功的 ChIP的95%的 的成S

37、YBR Green技術(shù)的qPCR的SYBR GreenTaqMan 的SYBR GreenTaqMan* ：的 MIQE MinimumInformationfor指的Ct*Ct 指的的CtDNA的的的DNACt的DNACt的 qPCRChIP的DNA儎的因儎Taq因 qPCRChIP DNA的 50 L ChIP2 L免 PCRPublication ofiveReal-TimePCR Experiments指南qPCR“”Cq “”Ct關(guān)從的 的 的 http:/ hp儎的T3&5的：http/articles/10-tips-for-consistent-real-time-pcr-rt

38、pcr/19ChIPqPCR亱K的的 : 的DNADNA的 技術(shù)的僂儎的 /的成+ 的3.4.1. qPCR的的qPCR的 亱 的俌 qPCR 的 EqPCR的的 的的的Ct：Ef ciency (E) =10-1/的% Ef ciency = (E-1) x 100的 95-105%的 僂的：的的的的人 的DNA的的染qPCR僂的DNA 的因DNA的染色質(zhì)的 DNA的染色質(zhì) 的2-5%染色質(zhì)K 的 qPCR的的的3. 的 Ct18-304. 的的因：a. 65-150 bpb. 勒的儎免DNA的染的的染的 UV的勒的Milli-Q 的203.4.2. qPCR的：Ct的DNACt的DNA：E

39、=2:ChIP DNA/DNA的 = 2 Ct(control DNA)-Ct(ChIP DNA)2-Ct DNAChIP DNA的DNA指的免疫沉淀的染色質(zhì)ChIPDNA的DNA的CtChIP DNA的成僚：1. 的DNA的2. 的的logqPCR的Ct 的的DNA1.ChIP DNA的Ct DNA 的CtDNA的CtChIP DNA的CtCt：Ct = CtChIP DNA (Ctinput-Log2 Input dilution factor)ChIP的：%Input = 2(-Ct normalized ChIP)的Ct的Ct 的CtCt： (Ct = Ctitive Ctnegati

40、ve)ChIP DNA的：DNA的qPCR的亱的僂Ct的的DNA的 DNA的“”2.3.的IgG免疫沉淀mock IP的的因的因僂的Western blot的“” 質(zhì)qRT-PCR的因cDNA 免疫沉淀質(zhì)的DNA 的因的DNA4.Fold enrient =2Ct1.2.Kuo, M.H., et al. Methods 1999; 3:425-433.Zhang, L., et al. Biochem. Bio ys. Res. Commun. 2004 ; 322: 705711.Livak, K.J., et al. Methods 2001; 25(4):402-4.6

41、.0.11.Sittgen, T.D., et al. Nat Protoc. 2008; 3(6):1101-1108.Das, P.M., et al. Biotechniques. 2004 ; 37(6):961-969. Collas, P. Methods Mol Biol. 2009; 567:1-25.Turner, F.B., et al. Methods Mol Biol. 2006; 325:261-272. Weann, A.S., et al. Methods. 2002; 26(1):37-47.Ngre, N., et al. Methods Enz

42、ymol. 2006; 410:316-341. Wells, J., et al. Methods. 2002; 26(1):48-56.Sambrook, J., et al. Cold Spring Harb Protoc; 2006;:10.1101/t4455.Sambrook, J., et al. Cold Spring Harb Protoc.2006;:10.1101/t4456.La m, J.A., et al. Nat Struct Mol Biol. 2007; 14(11):1017-1024.CtCt的PCR的100%的E = 2的的12.13.21從從4的僚/從

43、/“DNA”PCRPCR的PCR的/的ChIPDNAPCR伄免人的勒的Milli-Q的的ChIPqPCR僂的PCR的ChIP儎的的的2-10 g ChIP的質(zhì)的的 的的的ChIP22的ChIP亯/ChIP免疫僂的關(guān)的AbSurance 16-66516-66716-668儎ChIP的 僂/的免疫僂Western blot免疫免疫沉淀ChIPDNA的DNAChIP：ChIP僂的的ChIP Ab+關(guān)ChIP的com/ geneticsChIP的 ChIP：1-10 g ChIP的的的質(zhì)的的：的的的：5.3染色質(zhì)的僚 僚免：的ChIP的從的成僚：的1%僂的：的質(zhì)DNA偵 X-ChIP質(zhì)DNA的質(zhì)的：

44、G16-662的啖的A/G 16-663PCR：僂的的IgGmock IP的儎的：的的：人僚的染色質(zhì)的：200-1000 bp的染色質(zhì)的的染：勒染“DNA”的PCR的染從/的僚/ChIP染色質(zhì)的的 因的功從NC的的ChIP的ChIP的IgG因啖啖IgG的染色質(zhì)ChIP的染色質(zhì)的免的染色質(zhì)染色質(zhì) 的ChIPChIP1%的的10 人質(zhì)的1520 的的的vs.的4 kb的的成的PBS 僚的的ChIP DNA的&K,3僂染色質(zhì)勒的1%的15 染色質(zhì)Magna ChIP G Ti e Kit 17-20000的的僂ChIP的 的Farnham僂的23DNA的因1000“染色質(zhì)”的指的染色質(zhì)質(zhì) ChIP

45、DNA僚的的儎DNA的的K539480-100mg的DNA成功染色DNA的免疫沉淀的ChIP 的的染色質(zhì)的儎的SDS1%染色質(zhì)染色質(zhì)IP因的ChIP的A/G的A16-661G16-662AG的免的AG A/G16-663的的的質(zhì) 的的的儎的染的染色質(zhì)mock IP從/的僚/儎37C染色質(zhì) 染色質(zhì)15 mL0.6 mL1.2 mL的 PCR qPCRqPCR因的因的 qPCRqPCRCtqPCR的的IgG的%IgG pulldownqPCR的因的mock IgG的的成的ChIP的因的IgG的Ct 的ChIP的因的ChIP儎的IgG的的ChIP的qPCR僂的的的 的的qPCR僂的qPCRqPCR的

46、的的染染SYBR Green的24qPCRqPCR僂的的 的DNA的因-倉(cāng)的僂的DNA染SYBR Green的qPCRChIP DNA的的質(zhì)的儎儎的僂 IgG的5成功僂的的的 Ct IgG 的mock IPChIP IP的mock IPIgG的Ct的DNAChIP僂的DNAPCR的Ct ChIP的qPCRChIP9696ChIP儎唎 的Magna ChIP HT96 ChIP Kit 17-10077EZ-Magna ChIP HT96 Kit 17-10078的 的的亱的55.1. ChIP的5.2. ChIP的1.1.20 mL的150 mm的的勒的成1 mm儎FFPE的的80-90%的2

47、.150 mm22 mL 1X PBS2.2 mL 10X PBS19.8 mL人Magna ChIP G Ti e Kit的 17-20000 50 mL人的1X PBS20 mL人的PBS550 L 37%的1100 L 18.5%的0 mL550 L 37%的 1100 L 18.5%的.101X：的2ml人的1XPBS10 L 200XIII2 mL 10X的101X：的2 mL人的1X PBS10 L 200XIII2 mL 10X的Dounce4C800 x g5沉淀.的800 x g510 mL人的1X PBS1X PBS1

48、X PBS12. 2 mL5的1X III13. 的的 14. 4C800 x g5沉淀255.3.DNA的DNA成200-1000bp的的 功的：1 LK62C21-2%10 L20 L 100 bp DNA marker的免 200-1000 bp的DNA的DNA的 的 的染色質(zhì)免疫沉淀僂 的1.mL的的mL的 的/功A .B .C .5.4. DNA Sambrook, et al., 2006A亯的1.5.13的5X106 ml5X107 ml.的僚500 L 的的DNA4 的: 7.8-8.0的成的的80% 1 0.5 M10%儎 因 的8-哺色的 99

49、.5%儎DNA1050pg DNA 的成 倉(cāng)的沉淀僚DNA的的的PCRDNA1017-10451CpGenomeDirectPrepBisul teKit 50rxn17-10452CpGenomeDirectPrepBisul teKit 200rxn DNAU: CpG WIZ Unmethylated Primer M: CpG WIZ Methylated Primer W: CpG WIZ Wild Type Primer17-10454CpGenomeDirectPrep96Bisul teKit32New AntibodyDescriptionHostSpecies Reacti

50、vityKey ApplicationsCat. No.PUBLICATION HIGHLIGHTAnti-Histone H3.3,K27M mutant(Cat. No. ABE419)Anti-BANF1, clone EPR7669RabbitHuWB, IHCMABE537Anti-Bmi-1, clone EPR3745(2)RabbitHuWB, IHCMABE480Anti-BonzoRabbitHuWB, IHCABE604entrin-2RabbitHu, Ms, RtWB, IC, IHC, IPABE480entrin-3RabbitHu, Ms, RtWB, IP,

51、IC, IHCABE481In a recent Science pr titled: Inhibition of PRC2 activity by a gain-of- function H3 mu ion found in pediatric glioblastoma* the Allis laboratory and colleagues (representing Rockefeller, Princeton, Duke and the University of Pennsylvania) used Merck Millipores newly released anti-Histo

52、ne H3.3 K27 mutant, rabbit polyclonal antibody (Cat.No. ABE419) to exploregenetic controlHD-4, clone 3F2/4MouseHu, MsWB, IHC, ChIP, IFMABE455Anti-dimethyl G9a (Lys185)RabbitHuWB, PIA, IPABE489Anti-dimethyl Histone H3 (Arg17), symmetricMouseHuWB, DB, ICMABE246Anti-dimethyl Histone H3 (Lys36)RabbitHuW

53、B, DB, ChIP07-369-IAnti-FOXO4, clone EPR5442RabbitHuWB, FCMABE539Anti-H2A.Z.2.2, clone 1H11-11RatHuWBMABE347Anti-HIRA, clone EPR7416RabbitHuWB, IFMABE565Anti-Histone Deacetylase 6, clone EPR1698(2)RabbitHuWB, IHCMABE546in pediatric glioma and othathologies.Anti-Histone H1, clone 34MouseHu, Ms, Rt, B

54、ov, XenopusWB, IC, IHC, FCMABE446The authors discoveredt certain humangliomas containing the missense mu ion Lys27Met (K27M) in histone H3.3 displayed lower overall amounts of H3K27me3 both in vivo and in vitro. The Histone H3K27 mutant-speci c antibody from Merck Millipore was used in Western blott

55、ing,Anti-IDHRabbitHuWB, IHC(P)ABE454Anti-JMJD2DRabbitHu, ChimpWB, IHC, IPABE499Anti-KDM1BRabbitHu, MsWBABE495Anti-LSD1, clone EPR6825RabbitHu, Ms, RtWB, IHCMABE545immunohistochemistry and ChIPysisAnti-Mad1, clone BB3-8MouseHuWB, IFMABE867to reveal the expresof the gain-of-Anti-Mad2, clone AS55-A12Mo

56、useHuWB, IFMABE866function mu ion and correlate it to the disease s e. The researchers proeK-to-M substitutions may represent a mechanism to altergenetic s es in a range of pathologies.Anti-monoubiquitin H2A (Lys119)RabbitHu, MsWB, ChIP, ICABE569tAnti-Myb Binding Protein 1ARabbitHuWB, IC, ChIPABE577

57、Anti-PRabbitHuWB, ICABE606Anti- F19RabbitHuWB, IP, ChIPABE992Anti- os o-FOXO4 (Ser262), clone EPR2315(2)RabbitHuWBMABE555NOTE: To help you extend and complement these studies we have also just released aAnti-PTBP2RabbitHu, Ms, RtWB, IPABE431Anti-RAG-1RabbitHuWB, IC, IPABE440binant H3K27Mbinant prote

58、in.Anti-Sirt1(Sir2), clone 3-10RabbitMsWB, IHCMABE426See the the Proteins, Enzymes and ReagentsAnti-Ski, clone G8MouseHuWB, IHCMABE442Sectiothe next page.Anti-SPT5RabbitHuWB, IPABE443*Lewis PW et al. Science. 2013 May 17;340(6134):857-61.Anti-SUV39H1, clone EPR4960(2)RabbitHuWBMABE552Anti-TCF-4, clo

59、ne 5F9.1MouseHuWB, IHCMABE361Anti-TET2RabbitHuWBABE595LEGENDSpecies: Hu=Human, Ms=Mouse, Rt=Rat, Bov=Bovine, Mky=Monkey, Can=Canine, Prim=Primate, Por= Porcine, Chimp=Chimpanzee, Rhe Mac=Rhesus Macaque, Ham=Hamster, Chk=ChickenApplications: FC=Flow Cytometry, IC=Immunocytochemistry, IHC=Immunohistoc

60、hemistry, IHC(P)=Immunohistochemistry (Paraf n), IF=Immuno uorescence, IP=Immunoprecipi ion, WB=Western Blotting, ChIP=Chromatin IP,Anti-TET2, clone hT2H 21F11MouseHu, MsWB, IPMABE462Anti-P11RabbitHu, Ms, RtWB, IP, ChIP, IHCABE567Anti-THRAP3RabbitHuIP, IC, WBABE487Anti-TIF1-RabbitHu, MsWB, ICABE434A