成人的字典里没有“容易”二字,而做科研的人似乎对“不容易”有更深刻的理解。每做一个新的实验犹如西游记中唐憎取经路上的一次与妖魔鬼怪的搏斗,每一次实验的成功好似孙悟空降服了各路妖怪。作为初学者,一开始做常规的染色质免疫共沉淀(ChIP)实验,得到完好的DNA 需要耗时2天左右,而在这两天的实验过程中,我们要对ChIP 实验的每一步都再三斟酌,对染色质的质量做准确无误的质控后才能开始后续的IP与纯化等步骤。即使实验每一步都能顺利,但最终是否取得了真(zhen)阳性(jing),还需要多个指标来评价。
ChIP实验是通过抗体把DNA-Protein 的复合物沉淀下来,然后对与蛋白结合的 DNA 进行纯化, 随后,进一步对DNA 进行普通PCR, qPCR 或者测序等实验来分析结果。那如何来评价和计算ChIP-PCR的结果呢?
一般情况下,一次严格的ChIP 实验会设置阳性对照组、阴性对照组、目的蛋白组及input对照。我们通常看到普通PCR 的结果是图1这样的,这里研究了PPARγ与LPL (脂蛋白脂肪酶,lipoprotein lipase) 启动子区的结合情况。与染色质稳定结合的RNA poll-II 作为阳性对照,而ICAM-1是一个唯一定位于胞膜的蛋白,可以作为阴性对照。很多时候,阴性对照也可以是与目的抗体同源的IgG。
图1 普通PCR 结果示意图
但是CST推荐使用实时PCR(qPCR)来分析CIP 结果,因为它是定量的。因此,此文主要讲的是ChIP-qPCR 的设置、计算与评价。
关于 PCR 循环数
对于普通的PCR的单拷贝基因,我们建议使用大约30个PCR扩增循环; 对于多拷贝基因,如α卫星重复序列,我们建议使用大约20个PCR扩增循环,以确保PCR反应在线性扩增范围内。如果进行太多循环,则可以观察到Input样品,目的蛋白IP样品和IgG样品之间的差异较小,因为PCR扩增超出线性扩增范围并且所有信号变得饱和。 如果使用实时定量PCR(qPCR),则推荐40个循环的扩增,因为qPCR仪器能够实时记录每一循环的产物信号,通过还原完整扩增曲线来计算最初模板DNA的丰度。
ChIP-qPCR 的设置
ChIP-qPCR 的计算
计算CHIP-qPCR 富集效率时,一般是以相对Input信号富集比例来呈现的,例如若做了单个2%Input,计算公式如下:
注:CT 值一般是指2-3次重复实验的平均CT值
而 CST 一直推荐通过稀释不同比例的Input 来先建立qPCR 的标准曲线,标准曲线一般用来确定qPCR的反应效率。低扩增效率(小于90%)可能由于Taq酶抑制剂的污染、过高或未优化的退火温度、时间较久或已失活的Taq酶、引物设计不合理或扩增子含二级结构等。过高反应率(大于105%)一般是由于引物二聚体或者非特异性扩增。我们建议把Input 设置4个依次5倍稀释的点来确保标准曲线能覆盖实验中所有可能会用到的模版浓度(即形成宽的动态范围)。以总input染色质的2%开始,我们用这个稀释梯度系列(2%、0.4%、0.08%、0.016%)来为每个input染色质qPCR样品建CT的标准曲线。标准曲线是模版起始浓度的对数值对CT 值得到的,其后所得的线性回归直线方程,Pearson 相关系数(r)或决定系数(R2)可用来评价qPCR 反应是否需要优化。以下是如何制作标准曲线的方法及计算PCR 扩增效率的公式,可供参考。
注:Slope:标准曲线的斜率;Y intercept:标准曲线截距
这样我们就能通过将每个免疫沉淀样品的qPCR反应测出来的CT值转变为表示总input染色质的百分比的值,来计算每个免疫沉淀样品的DNA丰富度。
ChIP-qPCR 的评价
qPCR结果计算出来以后,我们还需要进一步比较阳性、阴性对照相对input富集丰度及目的信号相对于阳性、阴性对照富集丰度的倍数。经过成千上万次的ChIP-qPCR,CST 科学家给出了Q-PCR标准质控:
1)组蛋白H3阳性对照相对input富集丰度正常值在1%-5%左右;
2)阴性对照IgG相对input富集丰度应低于0.1%。
3)当靶标抗体在目标基因位点的相对富集丰度,相比于非目标基因位点(或阴性对照引物)的富集丰度比值超过3倍,相对于阴性对照IgG的相对富集丰度比值达到8到10倍以上时,ChIP结果信噪比达到合理范围,我们定义ChIP结果为阳性。
4)一般情况下,组蛋白相对于input富集丰度在1-50%范围;转录因子或辅因子相对于input富集丰度在0.2-1%。
具体如下:
真阳性的结果往往是图2这样的:
图2 真阳性ChIP–qPCR 结果
而假阳性的结果是图3这样的,主要的原因是IP 下来的DNA没有或不足 、PCR条件不合理等。
图3 忽悠人的假阳性结果如果想了解更多的ChIP 相关知识,可参考文末「猜你喜欢」。
请不要被“假阳性”蒙蔽了双眼。吃一堑,长一智。成千上万次的实验经验造就了金睛火眼,ChIP-qPCR唯有按CST 科学家严格的质控标准,才能检验出“真阳性”。
最后,CST 为所有ChIP验证的抗体提供阳性和阴性对照引物组,可点击
此处查询,或直接翻看下方表格。
需滑动以看完整表格
No.AntibodyReactivityPositive Control PrimersNegative Control Primers
14129AEBP2 (D7C6X) Rabbit mAbH, M, R, Mk, (Hm, C, B)7707 H, 5517 H
4486 H83200AhR (D5S6H) Rabbit mAbH
81126 H4486 H15103Aiolos (D1C1E) Rabbit mAbH, M
12899 H4486 H8486RUNX2 (D1H7) Rabbit mAbH, M, R, Mk
12376 H4486 H12556RUNX2 (D1L7F) Rabbit mAbH, M, R
12376 H4486 H33593ATF-3 (D2Y5W) Rabbit mAbH, M
66427 H4486 H11815ATF-4 (D4B8) Rabbit mAbH, M, R
13059 M7015 M14820ATRX (D1N2E) Rabbit mAbH
14901 H5516 H80775BACH2 (D3T3G) Rabbit mAbH
53538 H4486 H12120Bcl-11B (D6F1) XP® Rabbit mAbH, (M, R, Mk)
11951 H4486 H14895BCL6 (D4I2V) XP® Rabbit mAbH
12899 H4486 H5650BCL6 (D65C10) Rabbit mAbH, M12924 H, 12899 H
4486 H14020BMAL1 (D2L7G) Rabbit mAbH, M, R
13902 M7015 M6964Bmi1 (D20B7) XP® Rabbit mAbH, Mk7707 H, 5517 H
4486 H5856Bmi1 (D42B3) Rabbit mAbH, M, R, Mk7707 H, 5517 H
4486 H5848Brd2 (D89B4) Rabbit mAbH, M, (R, Mk)
62858 H4486 H13440BRD4 (E2A7X) Rabbit mAbH, (B, Dg, Pg)
12924 H4486 H11974RNF20 (D6E10) XP® Rabbit mAbH, M, R, Mk, (Hm, Dg, Pg, GP, Hr)5037 H, 4471 H
4486 H49360Brg1 (D1Q7F) Rabbit mAbH, M, R, Mk9673 H, 9702 H
4486 H11966BRM (D9E8B) XP® Rabbit mAbH, Mk, (Dg)9673 H, 9702 H
4486 H8814Non-phospho (Active) β-Catenin (Ser33/37/Thr41) (D13A1) Rabbit mAbH, M, R, Mk, (C, X, Z, B, Dg, Pg, GP, Hr)8973 H, 5111 H
4486 H8480β-Catenin (D10A8) XP® Rabbit mAbH, M, R, Mk, (Z, B, Pg, GP, Hr)8973 H, 5111 H
4486 H9581β-Catenin Antibody (Amino-terminal Antigen)H, M, R, Mk
5111 H4486 H9587β-Catenin Antibody (Carboxy-terminal Antigen)H, M, R, Mk, (C, X, B, Dg, Pg, Hr)8973 H, 5111 H
4486 H62184CBFβ (D4N2N) Rabbit mAbH, M, R, Mk
29965 M8986 M4771Acetyl-CBP (Lys1535)/p300 (Lys1499) AntibodyH, M, Mk, (R)
4829 H4486 H7389CBP (D6C5) Rabbit mAbH, M, R, Mk
4829 H4486 H7425CBP (D9B6) Rabbit mAbH, M, R, Mk
4829 H4486 H14696CBX8 (D2O8C) Rabbit mAbH, M, R, Mk, (Hm, B)7707 H, 5517 H
4486 H81464Cyclin T1 (D1B6G) Rabbit mAbH, M, R, Mk5516 H, 14905 H
4486 H4351CHD1 (D8C2) Rabbit mAbH, M, R, Mk, (B, Dg, Pg)4653 M, 7015 M
8985 M12011CHD4 (D4B7) Rabbit mAbH, M, R, Mk, (Hm, B, Pg, GP, Hr)5516 H, 7014 H
4490 H2895CHOP (L63F7) Mouse mAbH, M, R
13059 M7015 M14894COBRA1 (D6K9A) Rabbit mAbH, M, R, Mk, (Hm, B)
14905 H4486 H9198Phospho-CREB (Ser133) (87G3) Rabbit mAbH, M, R, (Z)
4829 H4486 H9197CREB (48H2) Rabbit mAbH, M, R, Mk, Dm
4829 H4486 H4820CREB (D76D11) Rabbit mAbH, M, R, Hm, Mk, Dm
4829 H4486 H3417CTCF (D1A7) XP® Rabbit mAbH, R, Mk, (B)
5172 H4486 H3418CTCF (D31H2) XP® Rabbit mAbH, M, R, Mk, (B, Hr)
5172 H4486 H2899CTCF AntibodyH, M, R, Mk
5172 H4486 H3742E2F-1 AntibodyH, (R, B)
7001 H4486 H14159EAF2 (E1R8C) Rabbit mAbH
14023 H4486 H4153EGR1 (15F7) Rabbit mAbH, M, R, (B)
5549 H4486 H4154EGR1 (44D5) Rabbit mAbH, M, R, (B)
5549 H4486 H14468ELL (D7N6U) Rabbit mAbH, M, R, (B, Dg, Hr)
14084 H4486 H13258Estrogen Receptor α (D6R2W) Rabbit mAbH9673 H, 9702 H
4486 H8644Estrogen Receptor α (D8H8) Rabbit mAbH
9702 H9673 H13826ERRα (E1G1J) Rabbit mAbH, M, R, Mk, (B, Dg, Pg, Hr)
13828 M8985 M14069ETS-1 (D8O8A) Rabbit mAbH, M, R4663 H, 14905 H
4486 H5246Ezh2 (D2C9) XP® Rabbit mAbH, M, R, Mk7707 H, 5517 H
4486 H4905Ezh2 AntibodyH, M, R, Pg7707 H, 5517 H
4486 H5348Phospho-c-Fos (Ser32) (D82C12) XP® Rabbit mAbH, M, R, (Hm, Mk, B, Pg, Hr)
7983 R7964 R2250c-Fos (9F6) Rabbit mAbH, M, R, (Hm, B, Pg)
7983 R7964 R2251FosB (5G4) Rabbit mAbH, M, R
7983 R7964 R4402FoxP1 (D35D10) XP® Rabbit mAbH, M, R, Mk12924 H, 12899 H
4486 H5841Phospho-FRA1 (Ser265) (D22B1) Rabbit mAbH, M, R, (Mk, B, Hr)
7983 R7964 R19967Fra2 (D2F1E) Rabbit mAbH, (B, Hr)
7983 R7964 R3306G9a/EHMT2 (C6H3) Rabbit mAbH, M, R, Mk, (B, Pg, Hr)
98428 H5516 H5852GATA-3 (D13C9) XP® Rabbit mAbH, M, (Mk)
13051 H4486 H5851GATA-6 (D61E4) XP® Rabbit mAbH, (M, R, Dg, Pg)
13840 H4486 H12041Glucocorticoid Receptor (D6H2L) XP® Rabbit mAbH, M, R, Mk
7681 H4486 H3660Glucocorticoid Receptor (D8H2) XP® Rabbit mAbH, M, R, Mk
7681 H4486 H41328Histone H1.4 (D4J5Q) Rabbit mAbH, Mk, (Hm, B, Dg)4486 H, 7014 H5037 H, 5516H
75336Acetyl-Histone H2AZ (Lys4/Lys7) (D3V1I) Rabbit mAbH, M, R
62086 H4486 H3724HA-Tag (C29F4) Rabbit mAball species expected
34589HDAC1 (D5C6U) XP® Rabbit mAbH, M, R, Mk5516 H, 7014 H
95064 H57156HDAC2 (D6S5P) Rabbit mAbH, M, R, Mk
7014 H95064 H85057HDAC3 (D2O1K) Rabbit mAbH, M, R, Mk9673 H, 9702 H
4486 H42427Helios (D8W4X) XP® Rabbit mAbH, M
99663 H4486 H36426Helios (D4Z6D) Rabbit mAbH
99663 H4486 H5537HIF-1β/ARNT (D28F3) XP® Rabbit mAbH, M, R, Mk
81126 H4486 H14179HIF-1α (D2U3T) Rabbit mAbH, M, R, Mk
75671 H4486 H8240Ubiquityl-Histone H2A (Lys119) (D27C4) XP® Rabbit mAbH, M, R, Mk7707 H, 5517 H4486 H, 5516H
12349Histone H2A (D6O3A) Rabbit mAbH, M, R, Mk, Z, (Hm, B, Dg, GP)4486 H, 5516 H, 4493 H, 7014 H
12799Acetyl-Histone H2B (Lys5) (D5H1S) XP® Rabbit mAbH, M, R, Mk, (Hm, C, Z, B, Hr)5516 H, 7014 H4486 H, 4490H
9072Acetyl-Histone H2B (Lys12) (D7H4) Rabbit mAb (ChIP Formulated)H, (M, R, Mk, C, X, Z, B, Pg, Hr)5516 H, 7014 H
9083Acetyl-Histone H2B (Lys15) (D8H1) XP® Rabbit mAbH, M, R, Mk, (Z, B, Pg, Hr)
7014 H4486 H5546Ubiquityl-Histone H2B (Lys120) (D11) XP® Rabbit mAbH, M, R, Mk5047 H, 4478 H5037 H, 4471H
12364Histone H2B (D2H6) Rabbit mAbH, M, R, Mk, (C, Z, B)4486 H, 5516 H, 4493 H, 7014 H
13576Phospho-Histone H3 (Thr3) (D5G1I) Rabbit mAbH, M, (R, Z, Sc)
4486 H5516 H53348Phospho-Histone H3 (Ser10) (D7N8E) XP® Rabbit mAbH, M, R, Mk, (Hm, X, Sc)5037 H, 4471 H
5326Mono-Methyl-Histone H3 (Lys4) (D1A9) XP® Rabbit mAbH, M, R, Mk, (Dm)
5047 H5037 H4473Pan-Methyl-Histone H3 (Lys9) (D54) XP® Rabbit mAbH, M, R, Mk, (C, Dm, X, Z, B, Pg, Sc, Ce)5098 H, 4486 H, 5077 H4471 H, 7014H
14186Mono-Methyl-Histone H3 (Lys9) (D1P5R) Rabbit mAbH, M, R, Mk, (X, Z, Pg, Sc, Hr)4478 H, 4490 H
4471 H14111Mono-Methyl-Histone H3 (Lys36) (D9J1D) Rabbit mAbH, M, R, Mk, (Hm, B)5516 H, 4490 H, 4493 H
5098 H12522Mono-Methyl-Histone H3 (Lys79) (D5X1S) Rabbit mAbH, M, R, Mk
4478 H4486 H9649Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAbH, M, R, Mk, Z, Ce, (Sc)5516 H, 7014 H, 7015 M4486 H, 4490H
9677Acetyl-Histone H3 (Lys9/Lys14) AntibodyH, M, R, Mk, Ce, (Z)7014 H, 7015 M4486 H, 4490H
7627Acetyl-Histone H3 (Lys14) (D4B9) Rabbit mAbH, M, R, Mk, (Hm, Dm, X, Z, Pg, Sc, Hr)7014 H, 7015 M4486 H, 4490H
13998Acetyl-Histone H3 (Lys18) (D8Z5H) Rabbit mAbH, M, R, Mk, Sc, (M, Hm, Pg)5516 H, 7014 H4486 H, 4490H
9675Acetyl-Histone H3 (Lys18) AntibodyH, M, R5516 H, 7014 H, 7015 M4486 H, 4490H
8173Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAbH, M, R, Mk, (Hm, X, Z, GP, Hr)5516 H, 7014 H, 7015 M5098 H, 4486H
4353Acetyl-Histone H3 (Lys27) AntibodyH, M, R, Mk, (Hm, C, X, Z, B)5516 H, 7014 H, 7015 M5098 H, 4486H
27683Acetyl-Histone H3 (Lys36) (D9T5Q) Rabbit mAbH, M, R, Mk, (Hm, Dm, X, Z, Pg)13653 H, 7014 H4486 H, 4493H
9725Di-Methyl-Histone H3 (Lys4) (C64G9) Rabbit mAbH, M, R, Mk7014 H, 7015 M4486 H, 4490H
4658Di-Methyl-Histone H3 (Lys9) (D85B4) XP® Rabbit mAbH, M, R, Mk, (Dm, X, Z, B, Pg, Sc, Ce)5098 H, 4486 H4471 H, 7014H
9753Di-Methyl-Histone H3 (Lys9) AntibodyH, M, R, Mk, Dm5098 H, 4486 H5516 H, 7014H
9728Di-Methyl-Histone H3 (Lys27) (D18C8) XP® Rabbit mAbH, M, R, Mk5098 H, 4490 H5516 H, 7014H
5427Di-Methyl-Histone H3 (Lys79) (D15E8) XP® Rabbit mAbH, M, R, Mk
5047 H5037 H9751Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAbH, M, R, Mk, Dm, Sc, (X, Z)5516 H, 7014 H, 7015 M4486 H, 4490H
9727Tri-Methyl-Histone H3 (Lys4) AntibodyH, M, R, Mk, (X, Z)5516 H, 7014 H, 7015 M4486 H, 4490H
13969Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAbH, M, R, Mk, (B)
4486 H5516 H5327Di/Tri-Methyl-Histone H3 (Lys9) (6F12) Mouse mAbH, M, R, Mk5098 H, 4486 H
5516 H9733Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAbH, M, R, Mk, (X, Z)4490 H, 4493 H
7014 H4909Tri-Methyl-Histone H3 (Lys36) (D5A7) XP® Rabbit mAbH, M, R, Mk, (Hm, C, Dm, X, Z, B)5047 H, 4478 H5037 H, 4471H
4620Histone H3 (D2B12) XP® Rabbit mAb (ChIP Formulated)H, M, (R, Hm, Mk, C, Dm, X, Z, B)4486 H, 7964 R, 7014 H, 7015 M
14269Histone H3 (1B1B2) Mouse mAbH, M, R, Mk4486 H, 5516 H, 8986 M, 4493 H, 7014 H, 7015 M
2650Histone H3 Antibody (ChIP Formulated)H, M, (R, Mk, C, Dm, X, Z, B, Hr)4486 H, 7014 H, 7015 M
8647Acetyl-Histone H4 (Lys5) (D12B3) Rabbit mAbH, M, R, Mk, (C, Dm, X, Z, B, Pg, Hr, Ce)
7014 H4486 H9672Acetyl-Histone H4 (Lys5) AntibodyH, M, R, Mk, (C, Dm, X, Z, B, Pg, Hr, Ce)
7014 H4486 H13944Acetyl-Histone H4 (Lys12) (D2W6O) Rabbit mAbH, R, Mk, (C, Dm)5516 H, 7014 H
4486 H13534Acetyl-Histone H4 (Lys16) (E2B8W) Rabbit mAbH, M, R, Mk5516 H, 7014 H
5737Tri-Methyl-Histone H4 (Lys20) (D84D2) Rabbit mAbH, M, R, Mk, (X, B, Pg)5098 H, 4486 H
5516 H14149Histone H4 (D2X4V) Rabbit mAb (ChIP Formulated)H, (M, R, Hm, Mk, C, B)4486 H, 4493 H
2616HP1α AntibodyH, M, R, Mk, (B)
12870 M12928 M8676HP1β (D2F2) XP® Rabbit mAbH, M, R, Mk, (Hm, B, GP)
12870 M12928 M12972HSF1 (D3L8I) Rabbit mAbH, M, R, Mk, B, Dg, Pg
5551 H4486 H4356HSF1 AntibodyH, M, R, Mk
5551 H4486 H9034Ikaros (D10E5) Rabbit mAbH
7273 H4486 H14859Ikaros (D6N9Y) Rabbit mAbH, M
7273 H4486 H76041IκB-ζ (D4I7C) Rabbit mAb (ChIP Preferred)M
39609 M8985 M13945INTS9 AntibodyH, M, R, Mk, (Hm, B, Dg, Pg)
14084 H4486 H4299IRF-4 (D43H10) Rabbit mAbH, R
5156 H4486 H4964IRF-4 AntibodyH
5156 H4486 H5628IRF-8 (D20D8) Rabbit mAbH, M, (R, Mk, X, B)
9013 H4486 H76684IRF-9 (D2T8M) Rabbit mAbH
57949 H4486 H15106IRF-4 (D9P5H) Rabbit mAbH, M, R
5156 H4486 H13594JARID2 (D6M9X) Rabbit mAbH, M, (Hr)7707 H, 5517 H
4486 H3270Phospho-c-Jun (Ser73) (D47G9) XP® Rabbit mAbH, M, R, Mk, Pg, (B)
7983 R7964 R9165c-Jun (60A8) Rabbit mAbH, M, R, Mk
7983 R7964 R3753JunB (C37F9) Rabbit mAbH, M, R, Mk
7983 R7964 R76010LEF1 (D6J2W) Rabbit mAbH
8973 H4486 H2184LSD1 (C69G12) Rabbit mAbH, M, R, Mk
14271 H4486 H2139LSD1 AntibodyH, M, R, Mk
14271 H4486 H9814Acetylated-Lysine (Ac-K2-100) MultiMab™ Rabbit mAb mixall species expected4471 H, 5516 H4486 H, 4493H
9441Acetylated-Lysine Antibodyall species expected5516 H, 7014 H, 7015 M4490 H, 4493H
11959MAML1 (D3E9) Rabbit mAbH, M, R, Mk
7273 H4486 H12166MAML1 (D3K7B) Rabbit mAbH, M, R
7273 H4486 H99169MBD3 (D1B8F) Rabbit mAbH, M, R, Mk
5516 H95064 H3619MCM2 (D7G11) XP® Rabbit mAbH, M, R, Mk
5148 H4486 H14950MED26 (D4B1X) Rabbit mAbH14023 H, 14905 H
4486 H97800MITF (D3B4T) Rabbit mAbH, M, Mk
12970 H4486 H12590MITF (D5G7V) Rabbit mAbH, M, R, Hm, Mk
12970 H4486 H14893MLLT1/ENL (D9M4B) Rabbit mAbH
14909 H4486 H85570MLX (D8G6W) Rabbit mAbH, M, R, Mk
95288 M53559 M5415Mouse (G3A1) mAb IgG1 Isotype Control5646MTA1 (D17G10) Rabbit mAbH, M, R, Mk
7014 H95064 H13987c-Myc (D3N8F) Rabbit mAbH, M, R, Mk
4779 H4486 H9402c-Myc AntibodyH, M, R, (Pg)
4779 H4486 H84406N-Myc (D1V2A) Rabbit mAbH, M
90678 H4486 H51705N-Myc (D4B2Y) Rabbit mAbH, M, (R)
90678 H4486 H58418MYST2 (D4N3F) Rabbit mAbH, M, R, Mk7707 H, 14909 H
4486 H8822Nanog (D2A3) XP® Rabbit mAb (Mouse Specific)M4653 M, 4659 M
7015 M5232Nanog (D73G4) XP® Rabbit mAb (ChIP Formulated)H4641 H, 4649 H
4486 H8785Nanog (D1G10) Rabbit mAb (Mouse Specific; ChIP Formulated)M4653 M, 4659 M
7015 M3580Nanog AntibodyH4641 H, 4649 H
4486 H4373NeuroD (D35G2) Rabbit mAbH, M, R
5549 H4486 H12540NF-κB1 p105/p50 (D7H5M) Rabbit mAbH, M, (Mk)
5552 H4486 H3035NF-κB1 p105/p50 AntibodyH, Mk
5552 H4486 H13586NF-κB1 p105/p50 (D4P4D) Rabbit mAbH, M, R
5552 H4486 H8242NF-κB p65 (D14E12) XP® Rabbit mAbH, M, R, Hm, Mk, Dg
5552 H4486 H6956NF-κB p65 (L8F6) Mouse mAbH, M, R, Hm, Mk, Mi, B, Dg, Pg
5552 H4486 H37359NF-κB2 p100/p52 (D7A9K) Rabbit mAbH
5552 H4486 H3608Notch1 (D1E11) XP® Rabbit mAbH, M, R
7273 H4486 H4147Cleaved Notch1 (Val1744) (D3B8) Rabbit mAbH, M, R
7273 H4486 H46743NRF1 (D9K6P) Rabbit mAbH, M, R, Mk
26727 H4486 H12721NRF2 (D1Z9C) XP® Rabbit mAbH, M, Mk
12635 M7015 M8157Oct-1 (D7B6) Rabbit mAbH9673 H, 9702 H
4486 H5677Oct-4A (C30A3C1) Rabbit mAb (ChIP Formulated)H, M4641 H, 4653 M, 4649 H, 4659 M4486 H, 7015M
2890Oct-4A (C52G3) Rabbit mAbH4641 H, 4649 H
4486 H2750Oct-4 AntibodyH, (Mk)4641 H, 4649 H
4486 H83932Oct-4A (D6C8T) Rabbit mAb (Mouse Specific)M4653 M, 4659 M
7015 M9284Phospho-p53 (Ser15) AntibodyH, M, R, Mk, (Mi, B, Pg)
6449 H4486 H2527p53 (7F5) Rabbit mAbH, Mk
6449 H4486 H32532p53 (D2H9O) Rabbit mAb (Rodent Specific)M, R
76144 M7015 M2524p53 (1C12) Mouse mAbH, M, R, Hm, Mk
6449 H4486 H48818p53 (DO-7) Mouse mAbH
6449 H4486 H9282p53 AntibodyH, Mk
6449 H4486 H13109p63-α (D2K8X) XP® Rabbit mAbH
6449 H4486 H59019PAX8 (D2S2I) Rabbit mAbH, Mk, (R)
14927 H4486 H3378PCAF (C14G9) Rabbit mAbH, M, R, Mk, (B, Hr)
4829 H4486 H13768PHC1 (1F3F3) Mouse mAbH, M7707 H, 5517 H
4486 H24799POLR1A (D6S6S) Rabbit mAbH, M, Mk
14901 H13653 H, 4486H
13499Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAbH, M, R, Mk, (Hm, Dm, X, Z, B, Pg, Sc, Ce)13653 H, 13669 H
4486 H13546Phospho-Rpb1 CTD (Ser2/Ser5) (D1G3K) Rabbit mAbH, M, R, Mk, (Hm, Dm, X, Z, B, Pg, Sc, Ce)13653 H, 13669 H
4486 H26319Phospho-Rpb1 CTD (Thr4) (D7L9W) Rabbit mAbH, M, R, Mk
20508 H4486 H13523Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAbH, M, R, Mk, (Hm, Dm, X, B, Pg, Sc, Ce)13653 H, 13669 H
4486 H13780Phospho-Rpb1 CTD (Ser7) (E2B6W) Rabbit mAbH, M, R, Mk
13669 H4486 H14958Rpb1 NTD (D8L4Y) Rabbit mAbH, M, R, Mk, (Hm, B)5037 H, 5516 H5098 H, 4486H
2629Rpb1 CTD (4H8) Mouse mAbH, M, R, Mk, (Hm, Dm, Sc)4471 H, 5516 H4490 H, 4493H
12825POLR3A (D5Y2D) Rabbit mAbH, M, R, Mk, (B, Dg, GP)
12769 H4486 H12137Brn2/POU3F2 (D2C1L) Rabbit mAbH, M, R
12377 H4486 H12495PRMT4/CARM1 (3H2) Mouse mAbH, M, R, Mk12531 H, 9673 H, 9702 H
4486 H8757Progesterone Receptor A/B (D8Q2J) XP® Rabbit mAbH, (Mk)
7859 H4486 H2258PU.1 (9G7) Rabbit mAbH, M, (Pg)
4486 H2266PU.1 AntibodyH, M, (Mk, Pg)
4486 H3900Rabbit (DA1E) mAb IgG XP® Isotype Control2729Normal Rabbit IgG9313Rb (D20) Rabbit mAbH, M, Mk
7001 H4486 H9309Rb (4H1) Mouse mAbH, Mk, B, Pg
7001 H4486 H13610RBL2 (D9T7M) Rabbit mAbH, M, R, Mk
13707 M7015 M13171RBBP5 (D3I6P) Rabbit mAbH, M, R, Mk, (C, X, B, Hr)
11951 H4486 H5313RBPSUH (D10A4) XP® Rabbit mAbH, M, R, Mk
7273 H4486 H12659c-Rel (D5G1A) Rabbit mAb (ChIP Formulated)H, (R, Mk)
12281 H4486 H10544RelB (D7D7W) Rabbit mAbH, M, R
5552 H4486 H13418Rev-Erbα (E1Y6D) Rabbit mAbH, M, R
13413 H4486 H5694RING1B (D22F2) XP® Rabbit mAbH, M, R, Mk7707 H, 5517 H
4486 H74775Pontin/RUVBL1 (D1L8J) XP® Rabbit mAbH, M, R, Mk
48781 H4486 H61702SET1A (D3V9S) Rabbit mAbH13653 H, 4471 H
4486 H7691SIN3A (D1B7) Rabbit mAbH, M, R, Mk, (Pg, Hr)9673 H, 5516 H4486 H, 4490H
8056SIN3A (D9D6) Rabbit mAbH, Mk9673 H, 5516 H4486 H, 4490H
6944Smad1 (D59D7) XP® Rabbit mAbH, M, Mk, (X, B)
5139 H4486 H9743Smad1 AntibodyH, M, Mk
5139 H4486 H11971Phospho-Smad1 (Ser463/465)/ Smad5 (Ser463/465)/ Smad9 (Ser465/467) (D5B10) Rabbit mAb (ChIP formulated)H, (M, R, Mk)
5139 H4486 H5339Smad2 (D43B4) XP® Rabbit mAbH, M, R, Mk5139 H, 4669 H
4486 H8685Smad2/3 (D7G7) XP® Rabbit mAbH, M, R, Mk5139 H, 4669 H
4486 H5678Smad2/3 AntibodyH, M, R, Mk, (X)5139 H, 4669 H
4486 H9520Phospho-Smad3 (Ser423/425) (C25A9) Rabbit mAbH, M, R, (Mk, X, Z, B)5139 H, 4669 H
4486 H9523Smad3 (C67H9) Rabbit mAbH, M, R, Mk, (X, Z, B)5139 H, 4669 H
4486 H38454Smad4 (D3M6U) Rabbit mAbH, M, R, Mk, (Dg, GP, Hr)
5139 H4486 H12534Smad5 (D4G2) Rabbit mAbH, M, R, Mk
5139 H4486 H91735SMARCB1/BAF47 (D8M1X) Rabbit mAbH, M, R, Mk9673 H, 9702 H
4486 H11956SMARCC1/BAF155 (D7F8S) Rabbit mAbH, M, R, Mk9673 H, 9702 H
4486 H12760SMARCC2/BAF170 (D8O9V) Rabbit mAbH, M, R, Mk, (Dg, Pg, GP, Hr)9673 H, 9702 H
4486 H5024Sox2 (D6D9) XP® Rabbit mAb (ChIP Formulated)H, (Mk, B, Dg, Hr)4641 H, 4649 H
4486 H23064Sox2 (D9B8N) Rabbit mAbH, M4653 M, 4659 M
7015 M2748Sox2 AntibodyH, M, (R, Mk, B, Dg, Hr)4641 H, 4653 M, 4649 H, 4659 M4486 H, 7015M
9389SP1 (D4C3) Rabbit mAbH, Mk
7531 H4486 H12191SPT16 (D7I2K) Rabbit mAbH, M, R, Mk, (Hm, X, Z, B, Dg, GP, Hr)11953 H, 12010 H5549 H, 4663H
15616SPT6 (D6J9H) Rabbit mAbH, M, R, Mk5516 H, 14905 H
4486 H2191SRC-1 (128E7) Rabbit mAbH, M, R, Mk
25661 H95064 H20301SRC-1 (D1M3Y) Rabbit mAbH, M, R, Mk
25661 H95064 H2126SRC-3 (5E11) Rabbit mAbH, M, R, Mk
12531 H4486 H5147SRF (D71A9) XP® Rabbit mAbH, M, R, Mk, Pg
4663 H4486 H21792SS18 (D6I4Z) Rabbit mAbH, M, R, Mk, (Dg, Hr)9673 H, 9702 H
4486 H13421SSRP1 (E1Y8D) Rabbit mAbH, M, R, Mk, (C)11953 H, 12010 H, 4663 H
5549 H9167Phospho-Stat1 (Tyr701) (58D6) Rabbit mAbH, M
5148 H4486 H7649Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAbH, M, R, (Mk)
5148 H4486 H8826Phospho-Stat1 (Ser727) (D3B7) Rabbit mAbH, M, R, Mk, (B)
5148 H4486 H9177Phospho-Stat1 (Ser727) AntibodyH, M, R, Mk, (Mk, B)
5148 H4486 H14994Stat1 (D1K9Y) Rabbit mAbH, M, R, Mk
5148 H4486 H14995Stat1 (D4Y6Z) Rabbit mAbH, M, R
5148 H4486 H9172Stat1 AntibodyH, M, R, Mk, (B, Dg)
5148 H4486 H72604Stat2 (D9J7L) Rabbit mAbH, M
30101 H4486 H9145Phospho-Stat3 (Tyr705) (D3A7) XP® Rabbit mAbH, M, R, Mk, (Hm, B, Pg, Hr)
4663 H4486 H9131Phospho-Stat3 (Tyr705) AntibodyH, M, R, Mk, (C, B, Dg)
4663 H4486 H34911Phospho-Stat3 (Ser727) (D4X3C) Rabbit mAb (ChIP Specific)H, (M, R)
4663 H4486 H9134Phospho-Stat3 (Ser727) AntibodyH, M, R, (Hm, B, Dg, Pg, Hr)
4663 H4486 H8768Stat3α (D1A5) XP® Rabbit mAbH, M, R, Hm, Mk, (Pg)
4663 H4486 H4904Stat3 (79D7) Rabbit mAbH, M, R, Mk
4663 H4486 H12640Stat3 (D3Z2G) Rabbit mAbH, M, R, Mk, (B, Pg)
4663 H4486 H9139Stat3 (124H6) Mouse mAbH, M, R, Mk
4663 H4486 H4134Phospho-Stat4 (Tyr693) (D2E4) Rabbit mAbH, (M, R, Mk, Pg)
9014 H4486 H5267Phospho-Stat4 (Tyr693) AntibodyH, (M, R)
9014 H4486 H2653Stat4 (C46B10) Rabbit mAbH, M, R
9014 H4486 H9351Phospho-Stat5 (Tyr694) AntibodyH, M, (R, B)
5131 M7015 M94205Stat5 (D2O6Y) Rabbit mAbH, M, R
5131 M7015 M9363Stat5 AntibodyH, M, R, Mk
5131 M7015 M5397Stat6 (D3H4) Rabbit mAbH, M, R, (Mk, B, Dg, Pg)
7710 H4486 H12800STF-1 (D1Z2A) XP® Rabbit mAbH, M, R, (B)
12864 H4486 H64828SPT4 (D3P2W) Rabbit mAbH, M, R, Mk, (Hm, Sc)5516 H, 14905 H
4486 H3737SUZ12 (D39F6) XP® Rabbit mAbH, M, R, Mk, (Pg, Hr)7707 H, 5517 H
4486 H4883TAZ (V386) AntibodyH, M, R, (Hr)
14927 H14928 H74499TBL1XR1/TBLR1 (D4J9C) Rabbit mAbH, Mk8973 H, 5111 H
4486 H44059TBP (D5C9H) XP® Rabbit mAbH, M, R, Mk, (C, B)5037 H, 4471 H4486 H, 4493H
12578TBP (D5G7Y) Rabbit mAbH, Mk5037 H, 4471 H
4486 H13232T-bet/TBX21 (D6N8B) XP® Rabbit mAbH
13051 H4486 H2569TCF4/TCF7L2 (C48H11) Rabbit mAbH, (M, C)
5111 H4486 H2565TCF4/TCF7L2 (C9B9) Rabbit mAbH, (M, R)
5111 H4486 H92529TET2 (D9K3E) Rabbit mAb (Mouse Specific)M
89052 M8985 M37785TFEB (D2O7D) Rabbit mAbH
27131 H4486 H12265TH1L (D5G6W) Rabbit mAbH, M, R, Mk, (Hm, B, Dg, Hr)
12558 H4486 H12550Vitamin D3 Receptor (D2K6W) Rabbit mAbH, M, (Hm, B, Pg, Hr)
25661 H4486 H13105WDR5 (D9E1I) Rabbit mAbH, M, R, Mk
4641 H4486 H14074YAP (D8H1X) XP® Rabbit mAbH, M, R, Hm, Mk, (B, GP, Hr)
14927 H14928 H14910BRD7 (D9K2T) Rabbit mAb (ChIP Formulated)H12531 H, 9673 H
4486 H58613FoxA1/HNF3α (D7P9B) Rabbit mAbH, M, R, Mk
32784 H4486 H83700NKX3.1 (D2Y1A) XP® Rabbit mAbH
84846 H4486 H62370SMRT (D8D2L) Rabbit mAbH, Mk
25661 H4486 H18950TET2 (D6B9Y) Rabbit mAbH
25946 H4486 H订阅新闻
这些引物组是我们内部用于验证ChIP抗体的。阳性对照引物组扩增已显示含有指定组蛋白修饰或被指定转录因子结合的基因位置,而阴性对照引物组不扩增含指定修饰或结合因子的区域。请注意,在指定基因位点发现的组蛋白修饰和转录因子可以根据细胞背景而不同,因此一些对照引物组可能对指定的细胞类型和/或处理具有特异性,这取决于所研究的组蛋白修饰或转录因子。然而,其他对照引物组更通用,并且可以用作多种细胞类型中的多种抗体的对照。例如,RPL30和GAPDH是在大多数细胞类型中有活性的持家基因,因此,RPL30和GAPDH对照引物组可用作在大多数细胞类型中与转录激活相关的组蛋白修饰的阳性对照。类似地,α卫星重复对照引物
#4486 扩增组成型异染色质的区域,并且可以用作组蛋白修饰与任何细胞类型中的转录激活相关的转录因子的阴性对照。
【参考文献】
Yulia Haim, Tanya Tarnovscki,Dana Bashari, and Assaf Rudich. A chromatin immunoprecipitation (ChIP) protocol for use in whole human adipose tissue. Am J Physiol Endocrinol Metab 305: E1172–E1177, 2013.
【致谢】
特别感谢Cell Signaling Technology 美国研发科学家陈芳提供的实验数据!
-The End-
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