ChIP实验步骤-英文原版chip技术
染色质免疫沉淀技术（ChIP）实验流程（转）
染⾊质免疫沉淀技术（ChIP）实验流程（转）⼀. 实验前准备：1. 实验设计与分组2. 实验试剂、耗材、仪器准备3. 试剂盒：Pierce™ Agarose chip Kit⼆. 实验开展：(以哺乳动物贴壁细胞为例)A. 交联与细胞裂解1. ⽤15cm培养⽫培养细胞，细胞量达80%-90%，细胞数量约为1x107 个，待⽤。
以下步骤基于1次ChIP试验2. 交联：向每个含有培养液的培养⽫中，加⼊16%的甲醛，使甲醛终浓度为1%. 轻轻晃动培养⽫, 使混匀, 室温孵育10min. (交联时间很重要，过长影响ChIP结果，过短交联不完全，产⽣假阳性)3. 终⽌交联：向上述培养⽫中，加⼊10X的⽢氨酸溶液使其终浓度为1X的。
混匀，室温孵育5min。
4. 吸出培养⽫中含有甲醛-⽢氨酸的混合培养基。
⽤1倍体积预冷的PBS清洗细胞两次。
5. 在1ml预冷的PBS加10ul的Halt Cocktail。
然后将此混合液加⼊清洗后细胞中，再⽤细胞刮搜集细胞，将细胞悬浮液⽤移液器转移到1.5m的微管离⼼管中。
6. 将搜集的细胞于3000g离⼼5min。
除去PBS，将细胞沉淀物保存在-80°，或者直接进⾏⼀下步骤：酶解附：蛋⽩量检测：WBB. 酶解断裂染⾊质1. 准备好上述交联好的细胞。
如果是冻存的，需在冰上解冻。
2.加100ul含蛋⽩酶抑制剂的Lysis Buffer 1⾄细胞沉淀物中吹打混匀，涡漩离⼼管15s，置于冰上孵育10min;9000g离⼼3min，弃除上清。
3. 加0.25ul的Micrococcal Nuclease (ChIP级) (10 U/µL)，涡漩离⼼管，在37°⽔浴锅温浴15min，每5min 颠倒混匀;4. 加10µl的MNase stop 溶液终⽌反应，短暂涡漩混匀，冰上孵育5min。
5. 9000g离⼼5min，去上清，重新获得核酸复合物。
6. ⽤50µl的含(蛋⽩酶/磷酸蛋⽩酶)抑制剂的Lysis Buffer 2重悬核酸复合物，置于冰上15min，每5min涡旋15s。
ChIP实验流程整理
1、ChIP实验用的苗是正常光照条件下生长的四周大的苗子，取1.5克嫩苗组织，放入50ml 1%甲醛溶液中，抽真空交联。
2、用2.5ml 2M甘氨酸溶液停止交联反应。
3、水洗苗子数次，然后将苗用吸水纸吸干，液氮碾磨，然后用25ml提取缓冲液1重悬。
extraction buffer I0.4 M sucrose,10 mM Tris-HCl, pH 8,10 mM MgCl2,5mM b-mercaptoethanol,0.1mM phenylmethylsulfonyl fluoride [PMSF],1* protease inhibitor; Roche),4、用神奇滤器或者是金属筛过滤，然后4000rpm, 4℃离心20分钟5、用1ml提取缓冲液2，重悬沉淀物，14,000 rpm ，4℃离心10分钟。
extraction buffer II0.25 M sucrose,10 mM Tris-HCl, pH 8,10mMMgCl2,1%Triton X-100,5mM b-mercaptoethanol,0.1mM PMSF,1*protease inhibitor)6、用300ul提取缓冲液3，重悬沉淀物，14,000 rpm ，4℃离心60分钟。
extraction bufferIII1.7Msucrose,10mMTris-HCl, pH8,0.15%Triton X-100,2mMMgCl2,5mMb-mercaptoethanol,0.1mM PMSF,1*protease inhibitor)7、粗核提取物用200ul裂解缓冲液重悬，在冰浴上孵育10分钟，以充分裂解细胞。
8、超声处理，以剪切基因组DNA，使DNA大部分断裂成200-1000bp大小，如果能把大部分控制在400-800bp则更佳。
超声过程中请一定注意要保持样品处于冰浴中，并且处于较低温度。
超声剪切的效果在后续去交联后可以用常规的DNA琼脂糖凝胶电泳检测。
chip操作步骤
磁珠吸附法chip操作步骤及试剂配方1、每盘细胞（8ml培液），加入11×Fixation solution 800ul使得甲醛的终浓度为1%，fixation solution为现配，室温摇床10min。
(甲醛能有效的使蛋白质-蛋白质，蛋白质-DNA，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; 交联时间如果过长，细胞染色质难以用超声波破碎，影响ChIP结果，而且实验材料也容易在离心过程中丢失。
交联时间如果过短，则交联不完全，产生假阴性。
甲醛的交联反应可被加入的甘氨酸终止。
)2、终止交联：加1M甘氨酸1.26ml，使其终浓度为0.125。
室温摇床5min。
3、用冰冷的PBS 冲洗两次后，加适量pbs+pmsf，用细胞刷刮下细胞，4℃ 1000RPM离心5min。
4、倒去上清，加入1ml Scell Lysis Buffer，冰上放置10min，匀浆器匀浆后转入2ml Ep管中，4℃ 5000RPM离心5min.5、弃上清，300ul nuclei Lysis buffer，吹散沉淀。
6、socinate破碎：1min on off 30 10次左右，4℃ 13000RPM离心20min，琼脂糖胶电泳，亮带集中在1000bp左右的方可。
(以便暴露目标蛋白，利于抗体识别。
)7、分别收集20ul样品做input -20℃保存。
(Input是断裂后的基因组DNA，需要与沉淀后的样品DNA一起经过逆转交联，DNA纯化，以及最后的PCR或其他方法检测。
Input对照不仅可以验证染色质断裂的效果，还可以根据Input中的靶序列的含量以及染色质沉淀中的靶序列的含量，按照取样比例换算出ChIP的效率，所以Input对照是ChIP实验必不可少的步骤。
)8、用 Chip Diluiton Buffer 稀释样品10倍9、准备beads，用Pre-Blocking buffer for dynabeads 洗beads3次.(Protein A是一种金黄色葡萄球菌细胞壁蛋白质，能特异性地与人和哺乳动物抗体（主要是IgG）的Fc区结合)，最好实验前一天准备beads。
组织染色质免疫沉淀技术(chip)-步骤
Chip步骤组织裂解：1.新鲜组织。
切成1-3 mm3小块。
2.转移组织到50ML试管里。
加入10 ml of 1X PBS.3.加甲醛至终浓度为1%。
室温下转动15—20mins。
（10ul）4.加2.5 M Glycine至终浓度为0.125 M(终止交联)。
4°C下转动10mins。
（0.5ml）5.100 g, 4°C 离心样本5mins。
6.弃上清，取沉淀。
用45 ml 冰冻1X PBS和25 ml 冰冻1X PBS各洗一次。
离心弃上清。
7.再加入2 ml 冰冻1X PBS。
匀浆机裂解组织。
1000 rpm，4°C ，离心5 min。
弃上清。
8.细胞裂解液重悬细胞。
加入蛋白酶抑制剂PMSF (10 ul per ml), aprotinin (1 ul per ml) andleupeptin (1 ul per ml).冰上孵育10-15mins9.5,000 rpm ，4°C离心5分钟。
取沉淀10.细胞核裂解液重悬细胞加入（8）中的蛋白酶抑制剂。
冰上孵育10-20mins。
11.接下来就进去超声过程了。
（接下来第一天的5）第一天1.细胞中加入1%的甲醛，8ml的培养液加入216 ul的甲醛，37度十分钟。
2.配制含有蛋白酶抑制剂的PBS 20 ml和含有蛋白酶抑制剂的SDS溶液1ml3.将细胞拿出来，迅速的移除含甲醛的培养基，加入含蛋白酶抑制剂的PBS洗两遍。
胰酶消化20秒，加入含蛋白酶抑制剂的PBS 1ml。
用细胞刮刀把细胞刮下，收集到1.5ml的离心管里面。
4.4度2000rpm离心10min，弃上清液，加入200ul含蛋白酶抑制剂的ＳＤＳ溶液。
吹打重悬细胞，冰上孵育１０分钟。
5.超声切割ＤＮＡ，总切割时间４min30sec，超声10sec,间隙10sec。
6.4度13000rpm离心10min，转移上清液到一个新的2ml的离心管，弃沉淀。
ChIP原理及实验方法
ChIP原理及实验方法ChIP（Chromatin Immunoprecipitation）是一种用于研究蛋白质与DNA相互作用的实验方法。
该方法主要用于探究转录因子与染色质的相互作用、蛋白质修饰与基因表达调控之间的关系等。
下面将详细介绍ChIP的原理和实验步骤。
ChIP的原理：ChIP的基本原理是通过特异性抗体结合到目标蛋白质上，然后通过交联、裂解、免疫沉淀和DNA提取等步骤分离出与目标蛋白质结合的DNA片段。
通过对这些DNA片段的分析，可以了解目标蛋白质在染色质上的分布情况，从而揭示蛋白质与DNA相互作用的生物学功能。
ChIP的实验步骤：1.交联：将细胞或组织与甲醛交联，使蛋白质与DNA形成稳定的结合。
2.裂解：将交联的样品进行裂解，使细胞核和染色质释放出来。
3.免疫沉淀：将特异性抗体加入裂解的样品中，使其与目标蛋白质结合。
通过免疫沉淀，可以分离出与抗体结合的蛋白质-DNA复合物。
4.洗涤：通过洗涤去除非特异性结合的蛋白质和DNA。
5.解交联：通过高温或酶解去除交联，使DNA恢复原始状态。
6.DNA提取：将解交联后的样品进行DNA提取，获取与目标蛋白质结合的DNA片段。
7.PCR扩增：使用特异性引物对提取的DNA片段进行PCR扩增，以检测目标DNA片段的存在与否。
8.数据分析：通过测定PCR产物的数量，可以推断目标蛋白质在染色质上的结合情况。
ChIP的注意事项：1.选择合适的抗体：要确保选择的抗体具有高特异性和敏感性，以避免误差和背景信号。
2.优化实验条件：包括交联时间、裂解方法、洗涤条件等，以获得清晰的信号和较低的背景噪音。
3.正负对照组：需要设置正负对照组，以验证实验结果的可靠性。
4.数据分析：可以使用定量PCR、测序等方法对ChIP的结果进行定量和定性分析。
总结：ChIP是一种用于研究蛋白质与DNA相互作用的重要实验方法。
通过ChIP，可以了解转录因子在染色质上的定位、蛋白质修饰与基因表达调控之间的关系等。
chip基本实验步骤
基本实验步骤（1）收获细胞，加入适量细胞IP裂解缓冲液（含蛋白酶抑制剂），冰上或者4℃裂解30min, 12,000g离心30 min后取上清；（2）取少量裂解液以备Western blot分析，剩余裂解液将1μg相应的抗体和10-50 μl protein A/G-beads加入到细胞裂解液，4°C缓慢摇晃孵育过夜；（3）免疫沉淀反应后，在4°C 以3,000 g速度离心5 min,将proteinA/G-beads离心至管底；将上清小心吸去，protein A/G-beads用1ml裂解缓冲液洗3-4次；最后加入15μl的2×SDS 加样缓冲液，沸水煮10分钟；（4）SDS-PAGE, Western blotting或进行质谱分析。
一、样品处理：免疫沉淀实验成功与否，第一步处理样品非常关键。
免疫沉淀实验本质上是处于天然构象状态的抗原和抗体之间的反应，而样品处理的质量决定了抗原抗体反应中的抗原的质量，浓度以及抗原是否处于天然构象状态。
所以制备高质量的样品以用于后续的抗体-agarose beads孵育对免疫沉淀实验是否成功非常关键。
在这个环节中，除了要控制所有操作尽量在冰上或者4°完成外，最为关键的是裂解液的成份。
用于免疫沉淀实验的样品一般是原代培养细胞裂解液或者细胞系裂解液。
我们以常用的RIPA裂解液为例（主要含有pH7.4左右的离子缓冲液，接近生理浓度下的NaCl,一定比例的去垢剂和甘油以及各类蛋白酶抑制剂等）来说明其各主要成份的用途，进而帮助我们如何针对不同的实验目的和不同的蛋白质特性来选择最佳的裂解液。
a. 缓冲液：离子缓冲液常采用pH7.4的Hepes或者Tris-Cl。
b. NaCl浓度一般习惯用150 mM,这主要是因为150 mM接近生理浓度，不会破坏蛋白质之间的相互作用。
然而细胞内部的NaCl浓度并不是均一的，局部NaCl 的浓度可以低到50 mM,150 mM的NaCl有可能会破坏这个区域的蛋白质相互作用。
CHIP实验操作过程
CHIP实验操作过程（一）、细胞的甲醛交联与超声破碎。
1、取出1平皿细胞（10cm平皿），在9ml培养基中加入243ul 37％甲醛，使得甲醛的终浓度为1％。
2、37摄氏度孵育10min。
（此时准备1*PBS，加入蛋白酶抑制剂混合物5ul/ml）。
3、终止交联：加10*甘氨酸900ul，混匀后，在室温下放置5min即可，置冰上。
4、吸尽培养基，用冰冷的PBS清洗细胞2次。
5、加入含有蛋白酶抑制剂的PBS 2ml，细胞刮刀收集细胞于15ml离心管中。
预冷后4度，700g，5min收集细胞。
（此时准备SDS Lysis buffer，加入蛋白酶抑制剂混合物5ul/ml）。
6、倒去上清。
按照细胞量，加入SDS Lysis Buffer。
使得细胞终浓度为每200ul含2×106个细胞。
这样每100ul溶液含1×106个细胞。
再加入蛋白酶抑制剂复合物。
假设MCF7长满板为5×106个细胞。
本次细胞长得约为80％。
即为4×106个细胞。
因此每管加入400ul SDS Lysis Buffer，将2管混在一起，共800ul。
7、超声破碎：VCX750，25％功率，4.5S冲击，9S间隙（共18S*3+9S）。
共14次。
（破碎前可取10ul样品，用于琼脂糖电泳，冻存）（二）、除杂及抗体孵育。
8、超声破碎结束后，15.000g 4度离心10min，去除不溶物质。
留取300ul做实验，其余保存于－80度，最多可以保存2个月。
（可取10ul样品，用于琼脂糖电泳，冻存）300ul中，100ul加抗体（anti-STAT1）做为实验组；100ul加anti-RNA Polymerase II做为阳性对照组；100ul加Normal rabbit IgG作为阴性对照。
9、15ml离心管，300ul的超声破碎产物中，加入2.7ml ChIP Dilution Buffer和13.5ul的Protease Inhibitor Cocktail II，再加入180ul Protein G Agarose，4度颠转混匀1h。
CHIP实验步骤
CHIP-qPCR实验步骤一、实验材料●主要试剂●主要仪器及器材二、实验步骤1. 细胞交联：a. 用1ml PBS重悬细胞；b.加28ul 37%甲醛（终浓度为1%）进行交联，室温翻转孵育10min；c.加入10×甘氨酸至终浓度为1×；室温翻转孵育5min，终止交联；d.4℃，3000g，离心3min；弃上清液；e. 用1ml预冷的1X PBS重悬细胞，用3000g，4℃，5min离心，去上清。
f．重复e步骤一遍（此步骤沉淀可冻存于-80℃）；2. 胞核制备和染色质碎裂：a. 用200ul Membrane Extraction Buffer(使用前加入2ul蛋白酶/磷酸酶抑制剂)重悬细胞；冰上静置10min；b. 4℃，3000g，离心3min；弃上清液；c. 用200ul MNase Digestion Buffer （使用前加入0.2ul DTT）重悬细胞核;d. 加入0.2ul MNase ,吹打混匀；37℃水浴30min，期间5min混匀一次；e. 加入20μL MNase Stop Solution；混匀后冰上放置5min;f. 4℃，9000g，离心5min；弃上清液；g. 用100μL of 1X IP Dilution Buffer（使用前加入1ul蛋白酶/磷酸酶抑制剂）重悬沉淀；h. 冰上超声打断5次，每次2min。
i. 4℃,9000g离心5min;转移上清到新的EP管中3. 免疫沉淀：a. 取10ul上清作为input,-20℃冻存备用；b. 取90ul上清加入410ul X IP Dilution Buffer；阳性对照组加入10ul Anti-RNA Polymerase II Antibody，IP组加入10ug目的抗体；阴性对照组加入2μL IgG 。
c. 样本管放到翻转仪4℃翻转过夜孵育；d. 震荡混匀Protein A/G磁珠，并取20ul到每个实验组中；e. 将样品置于磁力架上，静置2min进行磁性分离，弃上清；f. 加入1ml IP Wash Buffer 1，并吹打混匀；置于翻转仪上，洗涤5min；g．将样品置于磁力架上，静置2min进行磁性分离，弃上清；h. 重复步骤f~g 3次；i. 加入1ml IP Wash Buffer 2，并吹打混匀；置于翻转仪上，洗涤5min；j. 将样品置于磁力架上，静置2min进行磁性分离，弃上清；4. 洗脱：a.加入150ul 1X IP Elution Buffer重悬磁珠，37℃震荡孵育30min；b.将样品置于磁力架上，静置2min进行磁性分离，将上清转移到一个新的EP管中；c. input样本中加入150ul 1X IP Elution Buffer；d.各样本中分别加入6μL NaCl(5M) 、2μL 蛋白酶K(20mg/Ml);e. 65℃孵育2h;f. 使用DNA 纯化离心柱从样品中纯化DNA。
ChIP实验步骤
第一天：（一）、细胞的甲醛交联与超声破碎。
1、取出1平皿细胞（10cm平皿），加入243 ul 37%甲醛，使得甲醛的终浓度为1%（培养基共有9ml）。
2、37摄氏度孵育10min。
3、终止交联：加甘氨酸至终浓度为0.125M。
450 ul 2.5M甘氨酸于平皿中。
混匀后，在室温下放置5min即可。
4、吸尽培养基，用冰冷的PBS清洗细胞2次。
5、细胞刮刀收集细胞于15ml离心管中（PBS依次为5ml，3ml和3ml）。
预冷后2000rpm 5min收集细胞。
6、倒去上清。
按照细胞量，加入SDS Lysis Buffer。
使得细胞终浓度为每200 ul含2×106个细胞。
这样每100ul溶液含1×106个细胞。
再加入蛋白酶抑制剂复合物。
假设MCF7长满板为5×106个细胞。
本次细胞长得约为80%。
即为4×106个细胞。
因此每管加入400ul SDS Lysis Buffer。
将2管混在一起，共8 00ul。
7、超声破碎：VCX750，25%功率，4.5S冲击，9S间隙。
共14次（二）、除杂及抗体哺育。
8、超声破碎结束后，10，000g 4oC离心10min。
去除不溶物质。
留取300ul做实验，其余保存于-80oC。
300ul中，100ul加抗体做为实验组；100ul不加抗体做为对照组；100ul加入4 ul5MNaCl（NaCl终浓度为0.2M），65oC处理3h解交联，跑电泳，检测超声破碎的效果。
9、在100ul的超声破碎产物中，加入900ulChIPDilutionBuffer和20ul的50×P IC。
再各加入60ulProteinAAgarose/SalmonSpermDNA。
4oC颠转混匀1h。
10、1h后，在4摄氏度静置10min沉淀，700rpm离心1min。
11、取上清。
各留取20ul做为input。
一管中加入1ul抗体，另一管中则不加抗体。
ChIP具体操作流程
ChIP具体操作流程ChIP（染色质免疫沉淀）是一种用于研究染色质蛋白质与DNA相互作用的实验方法。
下面是ChIP的具体操作流程：1.细胞或组织处理：a.培养或收集要研究的细胞或组织。
b.如有需要，对细胞或组织进行处理，如刺激、药物处理等。
2.交联：a.向培养细胞或组织中加入足量的交联剂，如甲醛，使染色质与蛋白质交联。
b.在室温下轻轻摇动培养细胞或组织，使交联剂充分混合。
3.停止交联：a.加入甘氨酸或甘氨酸盐酸盐来中和剩余的交联剂。
b.在室温下继续摇动培养细胞或组织。
4.细胞或组织裂解：a.用裂解缓冲液裂解交联细胞或组织，释放出染色质。
b.在冰上孵育细胞或组织，使细胞或组织裂解。
5.染色质切割：a.使用限制性内切酶或超声波将染色质切割成较小的片段。
b.在室温下孵育样品，使切割反应进行。
6.免疫沉淀：a.加入特异性抗体（抗目标蛋白）到裂解的细胞或组织中，与目标蛋白结合。
b.在4℃下孵育样品，使抗体与目标蛋白结合。
c.加入蛋白A/G琼脂糖磁珠，使其与抗体-蛋白质复合物结合。
d.在4℃下孵育样品，使磁珠与抗体-蛋白质复合物结合。
7.洗涤：a.使用洗涤缓冲液洗涤磁珠，去除非特异性结合的蛋白质和DNA。
b.重复洗涤步骤，至少洗涤3次。
8.去交联：a.加入适量的去交联缓冲液，去除染色质与蛋白质的交联。
b.在65℃下孵育样品，使染色质与蛋白质解交联。
9.DNA纯化：a.使用DNA提取试剂盒提取免疫沉淀后的DNA。
b.按照提取试剂盒的操作手册进行操作。
10.DNA定量和质检：a.使用紫外可见光谱仪或荧光光度计测量提取的DNA的浓度。
b.使用琼脂糖凝胶电泳检测DNA的大小和纯度。
11.DNA分析：a.使用PCR、实时荧光PCR、测序等方法对ChIP后的DNA进行分析。
b.根据需要选择适当的方法进行进一步的分析。
需要注意的是，不同实验室可能会有一些微小的差异或优化步骤，这些步骤可能根据具体实验要求而有所不同。
此外，ChIP实验是一个复杂的过程，需要仔细操作和严格控制实验条件，以确保结果的准确性和可重复性。
ChIP基本流程图
ChIP基本流程图ChIP（染色质免疫沉淀）是一种用于研究蛋白质与染色质相互作用的实验技术。
下面是ChIP的基本流程图，以便更好地了解该技术的原理和步骤。
1.交联：首先，将细胞或组织交联。
这一步骤的目的是固定蛋白质-DNA复合物，以便后续的实验处理。
交联通常通过添加交联剂（如甲醛）来完成。
2.细胞裂解：将交联的细胞或组织裂解，以释放细胞核。
这可以通过机械破碎、化学裂解或超声波处理来完成。
裂解的目的是将染色质解离为小片段，以便后续的免疫沉淀步骤。
3.DNA切割：使用特定的限制酶或酶切剂切割DNA。
这一步骤的目的是产生适当大小的DNA片段，以便后续的免疫沉淀和测序分析。
4.免疫沉淀：将特定抗体与要研究的蛋白质结合。
这些抗体通常是针对特定蛋白质的单克隆或多克隆抗体。
将抗体与它们所结合的蛋白质-DNA复合物一起孵育，以形成抗原-抗体复合物。
5.洗涤：使用缓冲液洗涤掉非特异性的蛋白质-DNA复合物，以去除非特异性结合的物质。
这一步骤的目的是减少背景噪音，提高实验的特异性。
6.反交联：通过加热或酶切等方法去除交联剂，以使蛋白质-DNA复合物解离。
这将使DNA片段从蛋白质中释放出来，以便后续的纯化和分析。
7.DNA纯化：纯化被免疫沉淀的DNA片段，以去除杂质。
这可以通过酚/氯仿提取、柱层析或磁珠纯化等方法完成。
8.DNA分析：对纯化的DNA片段进行进一步的分析。
这可以包括PCR扩增、测序、芯片分析或基因组定位等技术。
这些分析将帮助确定与特定蛋白质结合的DNA区域，并揭示蛋白质与基因调控之间的关系。
ChIP作为一种重要的基因组学技术，广泛应用于研究基因调控、表观遗传学和疾病发生机制等方面。
通过了解ChIP的基本流程，我们可以更好地理解该技术的原理和操作步骤，从而更好地利用它来解答科学问题。
ChIP操作步骤
ChIP操作步骤chip实验步骤第一部分：细胞的甲醛交联和超声波破碎1.交联前的准备工作（1）细胞准备：在特定条件下处理细胞，以确保目标基因的转录激活。
细胞密度应达到1×106个细胞/10cm培养皿。
（2）配制1％甲醛溶液：270μl37%甲醛加入10ml无血清细胞培养液中即可，应使用高质量的甲醛。
2.交联和超声波破碎优化超声条件，以保证目的细胞的染色质dna能被剪切为200bp～1000bp的片段，具体操作如下（以hepg2细胞为例）：（1）小心吸取细胞培养基，加入1%甲醛溶液10ml，37℃孵育10min，然后放在冰上5min，终止固定。
（2）小心尽量吸净培养液，用预冷的pbs3ml洗细胞两次。
（3）加入含有PMSF（10ul）的预冷PBS 1ml。
刮去细胞并将其转移到离心管中。
在4℃下以1200rpm（约3000g）离心5min。
小心地去除上清液以获得细胞沉淀（细胞沉淀可在-80℃下储存数月）（4）以200μlsds裂解缓冲液重悬细胞（含pmsf：2ul），冰上放置10min（5）冰上超声剪切染色质dna，使其成为200bp～1000bp的片段（10个脉冲，每个20s，脉冲间隔20s）（6）加入5mnacl8μl.65℃，4小时至隔夜以脱交联（7）琼脂糖凝胶电泳观察剪切效果（所获超声剪切物可于-80℃保存数月）。
第二部分：免疫共沉淀如果超声条件优化已经优化好，在步骤“（一）.2(5)”后，进行下列步骤。
对于阴性对照，采用下述步骤“5”所述。
连接到上面的2（6）1.4℃，13,000rpm离心10min，转移上清至2ml离心管中。
2.用含有蛋白酶抑制剂μl的芯片稀释缓冲液1800。
将200μl上清液稀释至2ml。
3.为了去除不与蛋白质A-琼脂糖特异性结合的分子，添加75μL蛋白质A/鲑鱼精子DNA琼脂,4℃旋转孵育30min。
在4.4℃下，以1000 rpm离心1分钟以沉淀琼脂，并将上清液转移到新的离心管中。
CHIP技术操作步骤
CHIP技术操作步骤1.将细胞接种到2个10 cm玻璃平皿内，加入10 mL 培养液进行培养；2.当细胞密度长至70-80%时，进行甲醛交联（首次进行交联时，消化细胞进行计数，使每个平皿内细胞数长至1x107个/板）；3.吸弃培养液，PBS洗涤细胞2次，最后一次PBS不要吸走，预留10 mLPBS液于平皿内；4.每个平皿内加入270 µL 37%甲醛，至终浓度为1%，37℃交联染色质10 min；5.倒掉上清，冰浴PBS洗板2次（PBS内含有PMSF 1mmoL/L，aprotinin1µg/mL）；6.加入5 mLPBS，刮下细胞，收集到15 mL离心管中进行离心，2000 rpm/3min，弃上清；7.加入含有蛋白酶抑制剂（PMSF，aprotinin）的SDS lysis buffer 900 µL，将细胞吹打均匀，分成3管；8.将细胞悬液置于冰上，进行超声；（Out put:5W；间隙10s，工作15s），将细胞内基因组打碎至500-1000 bp的小片段；9.取出80 µL超声产物作为In put组冻存于-70℃；10.加入9倍体积IP dilution buffer摇匀，加入100 µL protein A agarose/salmonsperm DNA混合液，同时加入蛋白酶抑制剂，4℃，3h；11.2000 rpm/3min，收集上清，加入抗体（稀释度一般为1：1000），4℃，过夜；12.再加入80 µL protein A agarose/salmon sperm DNA混合液，4℃，3 h；13.2000 rpm/5min，弃上清，收集protein A agarose/antibody/DNA复合物；14.分别用以下buffer各1 mL，依次洗涤；●TSI液1 mL洗一次；●TSII液1 mL洗一次；●Buffer III 1 mL洗一次；●TE液1 mL洗2次；每次3000 rpm/3min离心，收集沉淀；15.加入Extraction buffer 250 µL洗涤2次，平板摇床摇15 min，收集约500 µL洗脱液；16.取出In put管，以及15步骤所得洗脱液，每管加入20 µL NaCl 5 M/L，65℃，4-6 h反应去交联；17.加入10 µL 0.5 M/L EDTA，20 µL 1M/L Tris-HCl，2 µL 10 mg/mL,蛋白酶K，45℃反应1-2 h；18.酚-氯仿法提纯DNA；●加入450 µL饱和酚，5000 rpm/10min，取上清，重复一次；●取上清转移至新离心管中，加入450 µL（氯仿：异戊醇=24：1），重复一次；●取上清转移至新离心管中，加入1/10体积3 M/L乙酸钠pH=5.2，2.5倍体积无水乙醇，轻柔混匀，悬液静置于-20℃3个小时，离心10000rpm/15min；●70%乙醇洗涤1-2次，倒掉上清，空气中静置20min或者真空干燥5min；●每管加入30 µLRF water，-20℃保存；19.每次取1-2 µL模板，PCR反应，1.5%EB胶检测。
chip 的操作指南(中英文)
Fast chromatin immunoprecipitation assayJoel D.Nelson 1,2,Oleg Denisenko 2,Pavel Sova 2and Karol Bomsztyk 1,2,*1Molecular and Cellular Biology Program and 2UW Medicine Lake Union Research,University of Washington,Seattle,WA 98109,USAReceived November 15,2005;Revised and Accepted December 12,2005ABSTRACTChromatin immunoprecipitation (ChIP)is a widely used method to explore in vivo interactions between proteins and DNA.The ChIP assay takes several days to complete,involves several tube transfers and uses either phenol–chlorophorm or spin columns to purify DNA.The traditional ChIP method becomes a chal-lenge when handling multiple samples.We have developed an efficient and rapid Chelex resin-based ChIP procedure that dramatically reduces time of the assay and uses only a single tube to isolate PCR-ready DNA.This method greatly facilitates the probing of chromatin changes over many time points with several antibodies in one experiment.INTRODUCTIONChromatin is composed of DNA,proteins and RNA (1–3).Chromatin structure is dynamic,responds to extracellular sig-nals,and controls gene expression,cell division and DNA repair (3–5).Chromatin is one of the most intensely studied structures in biology and ChIP assays have proven to be a powerful means to investigate a host of DNA-dependent pro-cesses (3,6,7).Along with other techniques (8,9),Chromatin immunoprecipitation reveals an extraordinarily rich and dynamic chromatin environment (3,9).The traditional method has several limitations,it takes several days to complete,requires DNA precipitations and involves multiple tube transfers (6).It is becoming increasingly clear that chromatin is a very dynamic structure (1,3,8,10–13).Thus,the traditional method may not be suffi-cient enough to explore the rich environment of chromatin.In the traditional ChIP protocol (6),DNA extractions and precipitations,are not only time consuming and tedious but also introduce steps for potential sample loss and contam-ination.This becomes a bigger problem in experiments invol-ving multiple chromatin samples.We set out to simplify the standard ChIP protocol by eliminating the multiple tube transfers to purify DNA.MATERIALS AND METHODS Mammalian cellsRat mesangial cells were grown in 150mm plastic cell culture dishes in RPMI 1640media supplemented with 10%FBS,2mM glutamine,penicillin (100U/ml),streptomycin (0.01%)and humidified with 7/93%of CO 2/air gas mixture (14).Yeast strainsMedia used for the growth of Saccharomyces cerevisiae were previously described (15);cells were grown at 30 C.The strain used in this study was MAT a HML a HMRa ade2-101his3-D 200leu2-D 1lys2-801trp1D -1ura3-52(16).ReagentsAntibodies and blocking peptides.The antibody to the C-terminal peptide of K protein was raised in rabbits as described before (17).Anti-Sir2p serum was a gift from Jasper Rine,University of California,Berkeley (18).The other antibodies used,anti-RNA polymerase II,anti-histone H3and rabbit IgG were from Santa Cruz (cat#sc-899),Abcam (cat#1791),and Vector Laboratories (cat#I-1000),respectively.The blocking peptide to K protein antibody was previously described (17)and the blocking peptide to the RNA polymerase II antibody was obtained from Santa Cruz (cat#sc-899p).Chelex-100was purchased from BioRad (cat#142–1253)and proteinase K from Invitrogen (cat#25530–015).In vivo cross-linking and immunoprecipitationsCell cross-linking was done by adding 0.8ml of 37%formaldehyde to 20ml of overlaying media for 15min at RT,followed by the addition of glycine to a final concentration of 125mM (6).After cross-linking,cells were harvested and then washed twice with 10ml phosphate-buffered saline (PBS).Cells from one dish were lysed with 1.0ml IP buffer [150mM NaCl,5mM EDTA,1%Triton X-100,0.5%NP-40,50mM Tris–HCl (pH 7.5)and 0.5mM DTT]containing the following inhibitors;10m g/ml leupeptin,0.5mM phenyl-methlysulfonyl fluoride (PMSF),30mM p -nitrophenyl phos-phate,10mM NaF,0.1mM Na 3VO 4,0.1mM Na 2MoO 4and*To whom correspondence should be addressed at UW Medicine Lake Union,Box 358050,University of Washington,Seattle,WA 98109,USA.Tel:+12066167949;Fax:+12066168591;Email:karolb@ÓThe Author 2006.Published by Oxford University Press.All rights reserved.The online version of this article has been published under an open access ers are entitled to use,reproduce,disseminate,or display the open access version of this article for non-commercial purposes provided that:the original authorship is properly and fully attributed;the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given;if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated.For commercial re-use,please contact journals.permissions@Nucleic Acids Research,2006,Vol.34,No.1e2doi:10.1093/nar/gnj004Published online January 5, 2006 at Library of the Third School of Clinical Medical of Peking University on September 22, 2012/Downloaded from10mM b-glycerophosphate.After one wash with1.0ml IP buffer the pellet was resuspended in1ml IP buffer(containing all inhibitors)and sheared with a sonicator microprobe for 4rounds of15,1s pulses at output level7(Misonix3000). Sheared chromatin was cleared by centrifugation(10min at 17000g,Eppendorf5403),split into two0.5ml fractions,and was used immediately or stored atÀ70 C.After adding anti-body pre-incubated(30min at room temp)with or without blocking peptide,0.5ml of the sheared chromatin fraction was incubated in an ultrasonic water bath(15min,4 C)(Bronson 3510).Tubes were centrifuged(10min at17000g,Eppendorf 5403)and the supernatant was transferred to fresh tubes con-taining20m l of washed protein A beads(Pharmacia)(19).The slurry was rotated for45min(4 C)and then the beads were washedfive times with1ml cold IP buffer containing no inhibitors.Yeast chromatin was prepared as previously described(6).Isolation of DNA using conventionalphenol–chlorophorm methodThis procedure is based on previously described methods (6,20).Briefly,DNA is eluted twice from the protein A beads with250m l of elution buffer(1%SDS and0.1M NaHCO3)for15min with periodic vortexing at room tem-perature.Cross-linking is reversed by adding20m l of5M NaCl and incubating the eluate overnight at65 C.After add-ing5m g linear acrylamide,as a carrier(21),DNA is precipi-tated with1.0ml100%EtOH.The pellet is washed with1ml 70%EtOH,and then dissolved in100m l TE buffer(pH8.0). Proteins are digested by adding11m l10·proteinase K buffer [0.1M Tris(pH7.8),50mM EDTA and5%SDS]and1m l of 20m g/m l proteinase K at50 C for30min.DNA is extracted using phenol/chlorophorm and then chloroform,precipitated with ethanol and thefinal DNA pellet is dissolved in200m l TE buffer.Isolation of PCR-ready DNA using the new methodA total of100m l of10%Chelex(10g/100ml H2O)is added directly to the washed protein A beads and vortexed.After 10min boiling,the Chelex/protein A bead suspension is allowed to cool to room temperature.Proteinase K (100m g/ml)is then added and beads are incubated for 30min at55 C while shaking,followed by another round of boiling for10min.Suspension is centrifuged and super-natant is collected.The Chelex/protein A beads fraction is vortexed with another100m l water,centrifuged again,and thefirst and the second supernatants are combined.Eluate is used directly as a template in PCR and makes up to25%of the final reaction volume.Real-time PCRThe reaction mixture contained5m l2·SYBR Green PCR Master Mix(Applied Biosystems),2.5m l DNA template and 0.3m M primers(10m lfinal volume)in384-Well Optical Reaction Plate(Applied Biosystems).Amplification(three step,40cycles),data acquisition and analysis were done using the7900HT Real-Time PCR system and SDS Enterprise Database(Applied Biosystems).CalculationsFactor density from ChIP assays are expressed as a signal ratio, R,using the following formula,R¼exp2(CT mockÀCT specific), where CT mock and CT specific are mean threshold cycles of PCR done in triplicates on DNA samples from specific and mock immunoprecipitations.RESULTS AND DISCUSSIONMethod developmentChelex-100resin has been used previously for DNA extraction from forensic specimens(22).We reasoned that addition of Chelex resin to immunoprecipitated chromatin samples could facilitate DNA extraction.To test if the Chelex-based method can efficiently extract DNA from immunoprecipitated chro-matin we used antibodies to hnRNP K(K protein).K protein is a conserved DNA/RNA-binding protein involved in gene expression including transcription(23–27).Using the tradi-tional ChIP assay,we have previously shown the binding of K protein to multiple gene loci(20).Recruitment of this factor to DNA was estimated by comparing the DNA signal obtained with the specific antibody to that obtained in mock immuno-precipitation where the antibody is blocked with a specific peptide(20).Control experiment(western blot,Figure1A) shows that K protein immunoprecipitation is blocked with the specific peptide.Sheared chromatin from3H-thymidine-labeled cells was pulled down with protein A beads and anti-K protein antibody pre-incubated with or without blocking pep-tide.Chelex-100suspension was added to the washed protein A immunoprecipitates and after boiling,the Chelex/protein A bead suspensions were treated with proteinase K.3H counts in the bead and supernatant fractions were measured by liquid scintillation.As shown in Figure1B most of the3H counts were recovered in the supernatants indicating that the DNA is efficiently extracted from the immunoprecipitated chromatin. In agreement with previous studies(20),these results also show that a fraction of chromatin binds to the antibody-loaded protein A beads non-specifically.The DNA eluted from protein A beads is typically treated with proteinase K to remove associated proteins.After proteo-lysis DNA is purified to remove the proteinase and peptide fragments(6).To avoid this DNA purification step,after treat-ment with proteinase K,we boiled the Chelex/protein A bead suspension containing genomic DNA.After centrifugation, the supernatant was used as template in real-time PCR with primers to the egr-1and b-globin genes.Figure1C illustrates that this procedure eliminated the potential inhibitory effects of proteinase K on PCR.We next estimated the concentration of proteinase K needed to isolate DNA from the chromatin precipitated with antibod-ies to hnRNP K(Figure1D).We found that DNA can be extracted even without proteinase K,however,addition of the enzyme increases DNA yield.The enhancing effect of proteinase K digestion was greater for the silent b-globin locus(Figure1D,lower panel)compared with the active egr-1 locus(Figure1D,upper panel),with a2.4-compared with 1.5-fold increase in specific DNA yield,respectively.In sub-sequent experiments,we used100m g/ml concentration of proteinase K.In addition,we found that30min incubatione2Nucleic Acids Research,2006,Vol.34,No.1P AGE2OF7at Library of the Third School of Clinical Medical of Peking University on September 22, 2012/Downloaded fromwith proteinase K is as effective as 4h (data not shown).In subsequent experiments we used 30min proteinase K treatment.We also introduced an improvement in the immunoprecipi-tation step itself.In the ChIP assay,immunoprecipitation is typically carried out over several hours (6).We found that pre-incubating sheared chromatin with the antibody in an ultra-sonic water bath (15min at 4 C)first and then binding to protein A beads (45min at 4 C)is sufficient to immunopre-cipitate chromatin.This faster method for chromatin IP works well with different antibodies (20,28).Verification of the fast ChIP assay in mammalian cells To verify the new method,we examined kinetics of the recruit-ment of RNA polymerase II and K protein to the PMA-inducible ing the traditional ChIP assay,we have previously demonstrated transient recruitment of hnRNP K to the inducibly transcribed egr-1locus (20).The recruitment of RNA polymerase II is a highly regulated key step control-ling the rate of mRNA synthesis from target gene loci (4,29).Treatment of rat mesangial cells with mitogens potently acti-vates the immediate-early egr-1gene (20).We compared kinetics of PMA-induced recruitment of RNA polymerase II and K protein to the egr-1locus to the induction of egr-1mRNA measured by real-time RT–PCR.This comparison revealed that the kinetics of RNA polymerase II recruitment to the egr-1gene parallels the PMA-induced changes in the transcript (Figure 2A).The nearly identical changes in mRNA levels (Figure 2A,top panel,blue lines)and recruitment of RNA polymerase II (Figure 2,middle panel,blue lines)vali-date our ChIP protocol.Note reproducibility of the results of two independent ChIP is similar to that of two separate RT–PCR parison of the new (Figure 2A,middleFigure 1.A Chelex-100-based method to isolate PCR-ready DNA from immunoprecipitated chromatin.(A )Anti-K protein antibody (10m g)was pre-incubated with (+)or without (À)blocking peptide (4m g)(30min,RT)(17).Lysates were incubated with the antibodies,complexes were pulled down with protein A beads (IP),and after washing,proteins were eluted by boiling in SDS–PAGE loading buffer.Proteins were resolved by SDS–PAGE and,after transfer to PVDF membranes,immunostained (IS)with anti-K protein antibody.(B )Cells were labeled with 3H-thymidine overnight (10m Ci in 10ml media).After cross-linking with formaldehyde,cells were lysed and sonicated.Half of the sonicated chromatin was incubated with antibody blocked with the peptide [(+)blocking peptide]and the other half with antibody that was not blocked [(À)blocking peptide].After five washes with 1ml of IP buffer,Chelex was added and the mixture was boiled.After cooling,proteinase K (200m g/ml)was added and the tubes were incubated at 55 C for 60min,mix was again boiled and beads were centrifuged.3H counts in the supernatant and the beads were measured using a liquid scintillation counter.(C )Purified rat genomic DNA (Total DNA)was boiled with the Chelex/protein A beads suspension.After cooling to room temperature the suspension was treated with [(+)proteinase K]or without [(À)proteinase K]proteinase K and then the mix was boiled again for 10min.The suspension was centrifuged and the supernatant was used as a template in real-time PCR using primers to the egr-1and b -globin genes.Three step real-time PCR was run for 40cycles.Results are expressed as 40-CT,(Threshold Cycle,Applied Biosystems,ABI7900manual),which directly reflects levels of amplicons.(D )Sonicated chromatin was incubated with anti-K protein antibody as before.After five washes with 1ml of IP buffer,Chelex was added and the mixture was boiled.After cooling the mix was treated without or with proteinase K (100or 200m g)for 60min (55 C),suspension was boiled again and the released DNA was used as a template in real-time PCR.Plots show values mean ±SD n ¼3.P AGE 3OF7Nucleic Acids Research,2006,Vol.34,No.1e2at Library of the Third School of Clinical Medical of Peking University on September 22, 2012/Downloaded fromFigure 2.Verification of the new ChIP protocol.(A )Serum-deprived rat mesangial cells were treated with PMA (10À7M)for indicated time points.Whole cell RNA was used in RT with random hexamer primers.Real-time PCR was carried out with primers to either egr-1(exon 1)or LAMC1(exon 28)genes.Results normalized to b -actin mRNA are shown as fold induction,mean ±SD,two experiments,PCR done in triplicates (top panel).Serum-deprived mesangial cells were treated with PMA as above (top panel).After cross-linking with formaldehyde,cells were lysed,pelleted and sonicated.Chromatin IPs were prepared with either RNA polymerase II (4m g)(Middle panel)or K protein (10m g)(Bottom panel)antibody with or without blocking peptides (4m g).Equal amounts of chromatin fraction were used in the IPs.DNA purified with either the new (solid blue line)or the conventional (dotted blue line)ChIP protocols was used as a template in real-time PCR.The results are expressed as a ratio of the level of PCR products obtained without (À)and with (+)blocking peptide.The graphs show results from two independent IPs done with the new ChIP protocol and results of one representative experiment is shown for the traditional method.PCR was done in triplicates (middle and bottom panel).(B )New ChIP protocol was used to assesses PMA-induced kinetics of RNA polymerase II and K protein recruitment to the different regions (I–VII)along the LAMC1gene in rat mesangial cells.The graph with results of RT–PCR analysis of mRNA is shown in the right panel (V).Results are are shown as mean values of two independent experiments.Diagram above the graphs represents LAMC1transcribed (rectangle)and flanking regions (lines).The arrows point at the sites of the respective pair of primers (I and II are 20and 5kb 50to the start of transcription,respectively,III is the promoter region,IV is exon 2,V is exon 28and VI and VII are 5and 20kb 30to the end of the last exon,exon 28).(C )Comparison of the density of RNA polymerase II,K protein and histone H3in the 50flanking (I)and transcribed (II)regions of egr-1,and at the silent b -globin (III)locus.Equal aliquots of sheared chromatin were used in the new ChIP assay with either anti-H3(4m g),anti-RNA polymerase II or K antibodies.For H3ChIP,purified rabbit IgG fraction (4m g)was used as a mock IP control.Diagram above the graphs represents egr-1and b -globin genes (rectangle)and flanking regions (lines).The arrows represent the sites of the respective pair of primers (I–III).e2Nucleic Acids Research,2006,Vol.34,No.1P AGE 4OF7at Library of the Third School of Clinical Medical of Peking University on September 22, 2012/Downloaded frompanel,solid blue line)and the conventional(Figure2A,dotted blue line)ChIP protocols done on the same extracts reveals similar PMA-induced kinetics of RNA polymerase II recruit-ment to the egr-1locus.Both methods also revealed similar kinetics for K protein recruitment to this locus(Figure2A, lower panel).Thus,results obtained with the new and the traditional methods were very similar.In comparison to the rapid and robust activation of the short egr-1gene(3.8kb),induction of the long laminin g1(LAMC1) (128kb)gene is slow and of small magnitude(Figure2A, upper panel,red lines);2to3-fold induction for LAMC1 mRNA levels compared with30–50-fold induction for egr-1 transcript.Results of ChIP analysis show that recruitment of RNA polymerase II and K protein to the last exon of LAMC1 (exon28,primer V,diagram in Figure2B)is much lower than to the egr-1locus and parallels low levels of mRNA induction (Figure2A,compare the red and blue lines).We next examined the inducible recruitment of hnRNP K and RNA polymerase II to the long and weakly induced LAMC1gene in greater details(Figure2B).Results of the ChIP analysis revealed that there was PMA-inducible increase in hnRNP K and RNA polymerase II recruitment to the LAMC1gene and50and30flanking regions.The highest levels were observed in the promoter region with the density not only decreasing along the transcribed region but also exhibiting different kinetics(Figure2B).Likewise,in the case of the egr-1locus(Figure2A),at most of the examined LAMC1 sites the recruitment of K protein resembled but was not ident-ical to that of RNA polymerase II(II–VI).At the intergenic sites20kb50and30from the gene there were low constitutive and PMA-inducible levels of K protein with little or no RNA polymerase II detected.Thefinding here that K protein binding differs from that of RNA polymerase II is consistent with the notion that hnRNP K is involved not only in tran-scription but also in chromatin remodeling(24).The higher density of hnRNP K at regions that encode laminin g1protein (IV and V)than at the intergenic sites(I and VII)indicates preferential K protein recruitment to domains that include LAMC1open reading frame(ORF).The analysis of the LAMC1gene illustrates that the new method has a sufficient signal to noise ratio to study genes that exhibit low levels of induction.The ability to simultaneously monitor DNA-binding of sev-eral factors enhances chromatin studies.We used equal ali-quots of chromatin from the same time course experiment to compare density of RNA polymerase II,histone H3and K protein at50-flanking and transcribed region of e gr-1gene and the silenced b-globin locus(Figure2C).These experiments revealed that at zero time point the histone H3density was comparable at transcribed(Figure2C,upper panels,II)and non-transcribed(I)regions of egr-1and at the silent b-globin locus(III).In the transcribed region of egr-1(II)there was large PMA-inducible loss of H3-DNA contact associated with RNA polymerase II recruitment to this site(Figure2C,center top and middle panel).The kinetics of K protein recruitment (Figure2C,bottom panel)is similar but not identical to that of RNA polymerase II.In the intergenic region5kb50to the egr-1gene and at the silent b-globin locus RNA polymerase II was not detected(Figure2C,I and III)and the level of H3 decreased slightly or remained the same after PMA treatment (Figure2C,top panels I and III).As in the case of the regions flanking the LAMC1gene(Figure2B,I and VII)there were low levels of K protein(Figure2C,bottom panels I and III). Finding K protein in the intergenic regions is consistent with the previous suggestion that its binding is genome-wide(20). These data confirm previous observations of the inverse rela-tionship between the density of RNA polymerase II and histones(30–36).These results also further validate the new ChIP method and show the ability to simultaneously monitor DNA-binding of several factors.The presence of RNA polymerase II in the transcribed and promoter regions but not in the intergenic(Figure2B and C) domains and the differential loss of H3-DNA contact (Figure2C,top panels,I–III)confirms the specificity of the fast ChIP assay.The high reproducibility of the results obtained with the new ChIP method is illustrated by close kinetics of changes in the density of RNA polymerase II,histone H3and hnRNP K protein at egr-1locus observed in two separate experiments (Figure2C).Also,note that in these experiments(Figure2C) the kinetics of RNA polymerase II and hnRNP K protein recruitment to the transcribed region of egr-1is similar to the set of two other experiments shown in Figure2A. These experiments illustrate that the new method yields repro-ducible results and allows probing of multiple factors in chro-matin preps from one experiment.We use one15cm($107cells)dish to prepare chromatin sufficient to IP with one antibody,with and withoutblocking Figure3.Verification of the new ChIP assay in yeast.The new and traditional ChIP methods were used to assess recruitment of the yeast Sir2p to HMR and an adjacent genomic locus.(A)Diagram of the yeast HMR locus(37).Primers for PCR analysis were designed to the indicated regions.(B)Results of ChIP analysis with either antibodies to Sir2p or no antibodies(mock IP).IPs and DNA purification using either the new(blue)or traditional(purple)methods were done in parallel with equal amounts of yeast chromatin.Purified DNA samples were analyzed as above with real-time PCR.Results are expressed as signal ratios of anti-Sir2p IP to mock IP.PCR were done in triplicates,data represented as mean±SD.P AGE5OF7Nucleic Acids Research,2006,Vol.34,No.1e2at Library of the Third School of Clinical Medical of Peking University on September 22, 2012/Downloaded frompeptide.DNA purified with this method from one chromatin IP is sufficient for80–100PCR.Verification of the fast ChIP assay in yeastS.cerevisiae has proven to be a valuable system to study chro-matin processes(6)and many heterochromatin factors were initially characterized in yeast[reviewed in(37)].Among these factors,Sir2p is a very conserved protein that binds to all major silenced domains in the yeast nucleus,including HML and HMR mating type loci(37).We estimated the den-sity of Sir2at HMR and at a control locus that does not bind this protein using the new and traditional ChIP methods.Equal amounts of extracts and antibodies were used in IPs.The results of real-time PCR analysis demonstrate that both meth-ods reveal Sir2p recruitment to the silenced HMR region but not to the control locus(Figure3).These results demonstrate that the fast ChIP method can be also used to study chromatin processes in yeast.In summary,we have developed a simple and efficient ChIP assay that is fast,and allows using of multiple antibodies in one experiment to simultaneously process many samples. The new ChIP assay will greatly facilitate experiments designed to study chromatin dynamics from yeast to mammals.ACKNOWLEDGEMENTSWe thank Dr J.Rine for antibodies,and members of KB lab for valuable discussions of the method.This work was supported by NIH DK45978,GM45134and Juvenile Diabetes Research Foundation(K.B.).Funding to pay the Open Access publica-tion charges for this article was provided by NIH DK45978. Conflict of interest statement.None declared. 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ChIP实验流程整理
1、ChIP实验用的苗是正常光照条件下生长的四周大的苗子，取1.5克嫩苗组织，放入50ml 1%甲醛溶液中，抽真空交联。
2、用2.5ml 2M甘氨酸溶液停止交联反应。
3、水洗苗子数次，然后将苗用吸水纸吸干，液氮碾磨，然后用25ml提取缓冲液1重悬。
extraction buffer I0.4 M sucrose,10 mM Tris-HCl, pH 8,10 mM MgCl2,5mM b-mercaptoethanol,0.1mM phenylmethylsulfonyl fluoride [PMSF],1* protease inhibitor; Roche),4、用神奇滤器或者是金属筛过滤，然后4000rpm, 4℃离心20分钟5、用1ml提取缓冲液2，重悬沉淀物，14,000 rpm ，4℃离心10分钟。
extraction buffer II0.25 M sucrose,10 mM Tris-HCl, pH 8,10mMMgCl2,1%Triton X-100,5mM b-mercaptoethanol,0.1mM PMSF,1*protease inhibitor)6、用300ul提取缓冲液3，重悬沉淀物，14,000 rpm ，4℃离心60分钟。
extraction bufferIII1.7Msucrose,10mMTris-HCl, pH8,0.15%Triton X-100,2mMMgCl2,5mMb-mercaptoethanol,0.1mM PMSF,1*protease inhibitor)7、粗核提取物用200ul裂解缓冲液重悬，在冰浴上孵育10分钟，以充分裂解细胞。
8、超声处理，以剪切基因组DNA，使DNA大部分断裂成200-1000bp大小，如果能把大部分控制在400-800bp则更佳。
超声过程中请一定注意要保持样品处于冰浴中，并且处于较低温度。
超声剪切的效果在后续去交联后可以用常规的DNA琼脂糖凝胶电泳检测。
CHIP染色质免疫共沉淀实验 Protocol
CHIP染色质免疫共沉淀实验是一种在全基因组水平上研究蛋白质与DNA相互作用的技术方法。
其实验原理是基于抗原抗体反应的特异性，从而实现对DNA结合蛋白及其DNA靶标的富集。
实验所需试剂和耗材包括：细胞培养及提取试剂、生物素标记试剂盒、抗体、蛋白质A琼脂糖珠、Triton X-100、ECL显影液等。
实验仪器包括：二氧化碳培养箱、倒置显微镜、离心机、染色质免疫沉淀仪等。
实验准备工作的要点包括：首先，要确认所用试剂和耗材的型号和保质期；其次，要确保细胞株和抗体的选择合适；最后，准备好实验所需的仪器设备并调试至最佳状态。
实验方法主要包括以下步骤：1.将细胞进行培养并提取染色质。
2.在染色质中加入对应于一个特定组蛋白标记的生物抗体，并用Triton X-100将抗原抗体混合物进行稀释。
3.在混合物中加入蛋白质A琼脂糖珠，以便吸附多余的抗体和未结合的蛋白质。
4.用洗涤液洗涤沉淀物，去除未结合的蛋白质和抗体，最后用变性液洗脱DNA。
5.用电泳法和显影法检测提取出的DNA片段。
注意事项包括：要保持细胞生长状态良好，并确保抗原抗体反应的时间和温度准确适宜；在加入蛋白质A琼脂糖珠后，要充分混匀以避免影响实验结果；最后，要注意控制好电泳参数和显影条件以保证结果的准确性和可靠性。
常见问题及解决方法包括：如果抗原抗体反应不充分，可以尝试增加抗体浓度或延长反应时间；如果未结合的蛋白质不能被有效清除，可以尝试增加洗涤次数或更换洗涤液；如果电泳条带不清晰或出现异常，可以尝试调整电泳参数或更换电泳液。
总之，CHIP染色质免疫共沉淀实验是一种研究蛋白质与DNA相互作用的有效方法，需要注意保持细胞生长状态良好、准确控制抗原抗体反应条件、充分洗涤未结合的蛋白质等关键点。
同时，针对实验中可能遇到的问题，要积极采取相应的解决方法，以保证实验结果的准确性和可靠性。
CHIP实验流程图
CHIP实验流程图1.实验准备1.1准备实验所需的器材和试剂：包括CHIP试剂盒、离心管、PCR管、电泳仪等。
1.2清洗实验器材：使用去离子水和酒精对实验器材进行清洗和消毒。
1.3准备实验样本：从细胞培养物或组织中提取DNA，并进行纯化和浓缩。
1.4质检实验样本：使用比色法或紫外分光光度计检测DNA的质量和浓度。
1.5设计实验方案：根据实验目的和样本特点，设计合适的实验方案。
2.交叉连接试验2.1设计引物：根据需要扩增的目标序列，设计合适的引物。
2.2 制备PCR反应体系：将引物、模板DNA、Taq酶和其他试剂按照一定比例加入PCR管中。
2.3进行PCR扩增反应：使用PCR仪对PCR反应体系进行温度循环扩增。
2.4检测PCR产物：将PCR产物进行电泳分析，观察扩增结果。
2.5确认PCR产物：使用测序技术对PCR产物进行测序，确认扩增结果。
3.CHIP实验3.1 交叉连接反应：将DNA样本与蛋白质进行交叉连接反应，形成DNA-protein结合物。
3.2 细胞裂解：使用细胞裂解缓冲液将细胞裂解，释放DNA-protein结合物。
3.3 DNA纯化：使用离心管将DNA-protein结合物离心，将上清液中的DNA进行纯化。
3.4DNA修饰：将纯化后的DNA进行修饰，如去磷酸化或甲基化等。
3.5 免疫沉淀：使用特异性抗体对DNA-protein结合物进行免疫沉淀。
3.6洗涤和离心：对免疫沉淀产物进行洗涤和离心，去除非特异性结合物。
3.7 DNA解交联：对免疫沉淀产物进行加热处理，解除DNA-protein结合。
3.8DNA纯化：使用离心管将解交联产物离心，将上清液中的DNA进行纯化。
3.9 DNA测序：对纯化后的DNA进行测序，获取DNA-protein结合物的序列信息。
4.结果分析4.1数据处理：将测序数据进行质量控制和去除低质量序列。
4.2 数据比对：将测序数据与参考基因组进行比对，找到DNA-protein结合位点。
CHIP技术操作步骤
CHIP技术操作步骤CHIP（Chemical Identification Profile）技术是一种常用于药物研发、化学分析和化学生物学研究的分析技术。
它基于微流控技术，可以实现高通量的化学反应和分析。
以下是CHIP技术的操作步骤：1.设计和制造CHIP芯片：首先，根据实验需求设计和制造CHIP芯片。
CHIP芯片通常由透明的玻璃或聚合物材料制成，其表面经过特殊处理，可以固定化分析物质，如抗体、酶、核酸等。
2.洗涤和预处理CHIP芯片：在使用之前，需要对CHIP芯片进行洗涤和预处理，以去除可能存在的污染物，并使芯片表面更适合固定化分析物质。
3.固定化分析物质：将需要固定化的分析物质溶液加到CHIP芯片的特定区域，利用化学键或物理吸附的方法将分析物质固定在芯片表面上。
这个过程通常需要在适当的pH、温度和时间条件下进行。
4.样品处理：将待测样品处理成适合于CHIP芯片检测的形式。
这可能涉及样品的纯化、浓缩、稀释等步骤，以确保样品中目标物质的浓度在合适的范围内，并且不会影响后续的分析步骤。
5.样品加载：将经过处理的样品加载到CHIP芯片上，通常通过微流控系统控制样品的注入和流动。
在样品流过芯片表面时，目标分析物质与已固定化的分析物质发生特异性的结合反应。
6.控制实验条件：在实验过程中，需要控制一些实验条件来提高分析的准确性和灵敏度。
这包括温度、压力、流速、反应时间等。
适当的实验条件可以确保后续的分析步骤能够准确地进行并且结果可靠。
7.反应和检测：待测样品与固定化的分析物质发生特异性的结合反应后，可以使用不同的检测方法来检测和测量结合事件。
常用的检测方法包括荧光检测、生物传感器、质谱分析等。
根据实验需求，选择适当的检测方法来获得精确的分析结果。
8.数据分析和解释：将检测到的数据进行分析和解释。
通过比较样品与对照组的差异，可以获得目标物质的定量或定性信息。
根据实验设计，可以利用统计学方法来确定结果的可信度和显著性。
CHIP实验流程图
CHIP实验流程图（CST9002）第一天A体内交联，细胞核制备并用核酸酶S7消化染色质B.分析染色质的浓度和酶切情况（推荐步骤）C. 染色质免疫沉淀第二天D.漂洗免疫沉淀的染色质：ArrayE. 将染色质从抗体/蛋白G微球上洗脱并解交联F.用离心柱纯化DNA。
G. 实时定量PCR方法：建议：实验中使用带滤芯的枪头以尽可能减少污染的可能性。
试剂盒中所包含的对照引物特异性识别人或小鼠的RPL30基因，既可以用于常规PCR也可以用于实时定量PCR。
如果用户做的ChIP样品来源于其它他物种，建议客户设计合适的特异对照引物并优化PCR反应条件。
建议使用热启动Taq聚合酶以减少产生非特异PCR产物的风险。
PCR引物的选择很关键。
引物应尽可能按照下列标准来设计：引物长度: 24个核苷酸最佳Tm值：60°C最佳GC含量: 50%扩增片段长度：150-200 bp（用于常规PCR）80-160 bp （用于实时定量PCR）实时定量PCR方法：1.选与所用定量PCR仪配套的PCR管或板，做好标记。
注意加入阳性对照组蛋白H3样品组，阴性对照正常兔IgG样品组，以及监控DNA污染的不加DNA模板的空白组对照。
另外在加入反应体系前，将2%样品输入对照做一系列稀释（不稀释，1：5，1：25，1：125），据此可以产生标准曲线并测算PCR扩增效率。
2.在每个反应管或孔（板上）中加入2 μl相应DNA模板。
3.按下面配比配置PCR反应液总管，记住计算时多算两份以补偿分管时的体积损失。
配好后，向每个PCR管中加入18 μl反应液混合物。
试剂每个PCR反应（20 μl）所需体积无核酸酶的水 6 μl5 μM RPL30 引物 2 μlSYBR-Green反应体系10 μl4.按下面程序执行PCR：a.预变性95°C 3 分钟b.变性95°C 10 秒c.退火及延伸：60°C 30 秒d.重复第2及第3步，共40个循环5.用定量PCR仪自带的程序分析定量结果。
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一、ChIP实验步骤第一天：（一）、细胞的甲醛交联与超声破碎。
1、取出1平皿细胞（10cm平皿），加入243ul 37％甲醛，使得甲醛的终浓度为1％。
（培养基共有9ml）2、37摄氏度孵育10min。
3、终止交联：加甘氨酸至终浓度为0.125M。
450ul 2.5M甘氨酸于平皿中。
混匀后，在室温下放置5min即可。
4、吸尽培养基，用冰冷的PBS清洗细胞2次。
5、细胞刮刀收集细胞于15ml离心管中（PBS依次为5ml，3ml和3ml）。
预冷后2000rpm 5min 收集细胞。
6、倒去上清。
按照细胞量，加入SDS Lysis Buffer。
使得细胞终浓度为每200ul 含2×106个细胞。
这样每100ul溶液含1×106个细胞。
再加入蛋白酶抑制剂复合物。
假设MCF7长满板为5×106个细胞。
本次细胞长得约为80％。
即为4×106个细胞。
因此每管加入400ul SDS Lysis Buffer。
将2管混在一起，共800ul。
7、超声破碎：VCX750，25％功率，4.5S冲击，9S间隙。
共14次。
（二）、除杂及抗体哺育。
8、超声破碎结束后，10，000g 4度离心10min。
去除不溶物质。
留取300ul做实验，其余保存于－80度。
300ul中，100ul加抗体做为实验组；100ul不加抗体做为对照组；100ul加入4ul 5M NaCl （NaCl 终浓度为0.2M），65度处理3h解交联，跑电泳，检测超声破碎的效果。
9、在100ul的超声破碎产物中，加入900ul ChIP Dilution Buffer和20ul的50×PIC。
再各加入60ul Protein A Agarose/Salmon Sperm DNA。
4度颠转混匀1h。
10、1h后，在4度静置10min 沉淀，700rpm离心1min。
11、取上清。
各留取20ul做为input。
一管中加入1ul 抗体，另一管中则不加抗体。
4度颠转过夜。
（三）、检验超声破碎的效果。
取100ul超声破碎后产物，加入4ul 5M NaCl，65度处理2h解交联。
分出一半用酚/氯仿抽提。
电泳检测超声效果。
第二天：（一）、免疫复合物的沉淀及清洗。
12、孵育过夜后，每管中加入60ul Protein A Agarose/Salmon Sperm DNA。
4度颠转2h。
13、4度静置10min后，700rpm离心1min。
除去上清。
14、依次用下列溶液清洗沉淀复合物。
清洗的步骤：加入溶液，在4度颠转10min，4度静置10min沉淀，700rpm离心1min，除去上清。
洗涤溶液：a. low salt wash buffer----one wash b. high salt wash buffer-----one washc. LiCl wash buffer------one washd. TE buffer------two wash15、清洗完毕后，开始洗脱。
洗脱液的配方：100ul 10％SDS， 100ul 1M NaHCO3， 800ul ddH2O，共1ml。
每管加入250ul洗脱buffer，室温下颠转15min，静置离心后，收集上清。
重复洗涤一次。
最终的洗脱液为每管500ul。
16、解交联：每管中加入20ul 5M NaCl（NaCl终浓度为0.2M）。
混匀，65度解交联过夜。
第三天：（一）、DNA样品的回收17、解交联结束后，每管加入1ul RNaseA（MBI），37度孵育1h。
18、每管加入10ul 0.5M EDTA， 20ul 1M Tris.HCl（PH 6.5），2ul 10mg/ml 蛋白酶K。
45度处理2h。
19、DNA片段的回收―――omega胶回收试剂盒。
最终的样品溶于100ul ddH2O。
（二）、PCR分析二、技术总结（一）、关于细胞细胞的生长状态要好。
因为细胞的生长状态直接影响细胞内部的基因表达调控网络，也很有可能影响你所研究的TF与其靶Promoter 的结合。
一般细胞长到75％－80％比较好。
（二）、关于抗体！抗体是实验成败的致命因素之一！必须是IP级别的抗体，另外如果经济条件许可的话，尽量买大厂的抗体。
不推荐国产抗体和santa cruz的抗体，即使是IP级别的。
单抗与多抗的选择也需要仔细考虑。
两种抗体各有利弊。
单抗特异性强，背景低。
但是单抗有一个致命的弱点，就是识别位点单一，而在ChIP甲醛交联的过程中，很有可能该位点被其它蛋白或核酸结合而被封闭，导致单抗不能识别靶蛋白。
多抗虽然没有这个问题，但是多抗特异性较差，背景可能会偏高。
一般而言，如果没有十足把握（单抗的识别位点远离靶蛋白与核酸结合的区域），选择多抗比较稳妥一些。
（三）、关于交联与超声破碎！这一块的确是ChIP实验中比较难把握的部分。
交联的程度会影响到超声破碎的效果，交联的程度越高，超声破碎就越不易把基因组打碎成小片段。
交联不充分，只有一部分靶蛋白与其Promoter相结合，富集得到的Promoter的量不高，实验假阴性。
交联过充分，基因组上结合了太多的蛋白，对超声破碎造成障碍。
另外也会增加背景。
一般来讲，按照我的经验，交联条件取决于细胞类型。
不同的细胞系，交联的条件也不一样。
例如：NIH－3T3的交联条件是室温（25摄氏度）下15min，1％的甲醛浓度，而别的细胞系则可能完全不一样。
而超声破碎的条件，机器不一样，条件也不一样。
当然如果你有bioruptor 这样的神器，那么超声破碎对你而言就是小菜一碟了。
一般，理想的超声破碎得到的片段大小是200bp－1000bp。
但是200bp－2000bp的范围也是可以接受的。
（四）、关于操作希望尽可能的保持低温（4度）。
沉淀的时候可以先在4度放置一会，等它自然沉降一些，再超低转速（500rpm等）离心使其完全沉降。
虽然说明书上说ChIP实验的过程中有几个可以停顿的地方，我还是希望你能够连续把它做完，直到PCR结果出来为止。
尽量避免实验中不可预知的影响因素。
（五）、关于解交联虽然说明书上说4小时已经足够，但是我还是希望你可以解交联过夜。
因为在那样的环境里，DNA不会降解，过夜解交联更充分些。
只是不要忘记在EP管口封上封口膜。
（六）、关于DNA片段的回收需要注意的是：样品中SDS样品较高，普通的PCR产物回收试剂盒回收，很有可能会在最终的样品中混入SDS，影响PCR实验结果。
小Tip：过柱前，在样品中加入一定量的异丙醇，能有效的消除SDS沉淀二、ChIP是一项比较流行的研究转录因子（ transcription factor, TF）与启动子（promoter）相互结合的实验技术。
由于ChIP采用甲醛固定活细胞或者组织的方法，所以能比较真实的反映细胞内TF与Promoter的结合情况。
这个优势是EMSA这个体外研究核酸与蛋白相互结合的实验方法所不能比拟的。
当用甲醛处理时，相互靠近的蛋白与蛋白，蛋白与核酸（DNA或RNA）之间会产生共价键。
细胞内，当TF与Promoter相互结合（生物意义上的结合）时，它们必然靠的比较近，或者契合在一起，这个时候用甲醛处理，能使它们之间产生共价键。
一般ChIP的流程是：甲醛处理细胞——收集细胞，超声破碎——加入目的蛋白的抗体，与靶蛋白-DNA复合物相互结合——加入Protein A，结合抗体-靶蛋白-DNA复合物，并沉淀——对沉淀下来的复合物进行清洗，除去一些非特异性结合——洗脱，得到富集的靶蛋白-DNA复合物——解交联，纯化富集的DNA片断——PCR分析。
在PCR分析这一块，比较传统的做法是半定量－PCR。
但是现在随着荧光定量PCR的普及，大家也越来越倾向于Q－PCR了。
此外还有一些由ChIP衍生出来的方法。
例如RIP（其实就是用ChIP的方法研究细胞内蛋白与RNA的相互结合，具体方法和ChIP差不多，只是实验过程中要注意防止RNase，最后分析的时候需要先将RNA逆转录成为cDNA）；还有ChIP-chip （其实就是ChIP富集得到的DNA片段，拿去做芯片分析，做法在ChIP的基础上有所改变，不同的公司有不同的做法，要根据公司的要求来准备样品）。
ChIP assay: Chromatin immunoprecipitation assay was performed according to the protocol of Ch IP assay kit (Upstate Biotechnology, Lake Placid, NY).1, For cells cultured in 2D, about 1-2X107 S1 cells were growth in 100mm dish, and were cross-lin ked by adding formaldehyde to final concentration of 1% and incubated in room temperature for 10 minutes. For cells growth in 3D, cells were isolated from EHS using PBS/EDTA, and also cross-li nked as cells growth in 2D.2, Wash cells twice using ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl f luoride (PMSF), 1microgram/ml aprotinin and 1microgram/ml pepstatin A). Note: Add protease in hibitors to PBS just prior to use. PMSF has a half-life of approximately 30 minutes in aqueous solu tions.3, Scrape cells into conical tube, Pellet cells for 4 minutes at 2000 rpm at 4℃.4, Cells were washed with PBS and resuspended in ChIP lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH8.0) add protease inhibitors (inhibitors: 1mM PMSF, 1microgram/ml aprotinin and 1m icrogram/ml pepstatin A).. After incubated 10 minutes on ice, cells were sonicated to shear DNA t o lengths between 200 and 1000 basepairs being sure to keep samples ice cold.5, Centrifuge samples (part A, step 7) for 10 minutes at 13,000 rpm at 4℃, and detected the OD2 60 of the lysates.6, Sonicated lysates were then diluted to OD260 2 with ChIP dilution buffer (). 60 ul protein A-aga rose beads (Upstate Catalog # 16-157) was added to sonicated lysates and rotated at 4 for one ho ur to reduce the non-specific dinding.7, Pellet agarose by brief centrifugation and collect the supernatant fraction, 20ul of lystates were taken out as input control.9, Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2ml superna tant fraction and incubate 2h at 4℃ with rotation. For a negative control, perform a no-antibody i mmunoprecipitation by incubating the supernatant fraction with 60 microliters of Salmon Sperm DNA/Protein A Agarose Slurry for one hour at 4℃ with rotation.10, Pellet agarose by gentle centrifugation (700 to 1000 rpm at 4℃, ~1min). Carefully remove the supernatant that contains unbound, non-specific DNA. Wash the protein A agarose/antibody/hist one complex for 3-5 minutes on a rotating platform with 1ml of each of the buffers listed in the o rder as given below:Low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 150 mM Na Cl); High salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 500 mM NaCl); LiCl buffer (0.25 M LiCl, 1% NP-40, 1% SDC, 1 mM EDTA, 10 mM Tris-HCl pH8.0); TE buffe r (20 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0).11, Elute the histone complex from the antibody by adding 250 microliter elution buffer (1%SDS, 0.1M NaHCO3) to the pelleted protein A agarose/antibody/histone complex from step 10 above.Vortex briefly to mix and incubate at room temperature for 15 minutes with rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to another tube and repeat eluti on. Combine eluates (total volume=approximately 500 microliters.)12, Add 20 microliters 5M NaCl to the combined eluates (500 microliters) and reverse histone-DN A crosslinks by heating at 65℃ for 4 hours.Note: Include the input DNA from this step.13, Add 10 microliters of 0.5M EDTA, 20 microliters 1M Tris-HCl, pH 6.5 and 2 microliters of 10mg /ml Proteinase K to the combined eluates and incubate for one hour at 45℃.14, Recover DNA by phenol/chloroform extraction and ethanol precipitation. Addition of an inert carrier, such as 20 microgramsglycogen, helps visualize the DNA pellet. Wash pellets with 70% ethanol and air dry.15, Resuspend pellets in an appropriate buffer for PCR or slot-blot reactions. PCR or slot-blot con ditions must be determined empirically. （本文来源于爱番茄 , 原文地址： /archives/399.html ）染色质免疫沉淀（ChIP）实验中应注意的细节问题仅供参考使用，最好实验条件需要自己摸索：1、cell counting：尽量做到准确，会影响input结果。