iCLIP - 全转录组,蛋白质与RNA相互作用的映射与个别核苷酸分辨率

Published 4/30/2011
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Summary

成绩单的RNA结合蛋白的空间安排,是一种转录后调控的关键因素。因此,我们制定个人核苷酸分辨率的紫外光交联和免疫(iCLIP),允许精确的全基因组RNA结合蛋白的结合位点的映射。

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Konig, J., Zarnack, K., Rot, G., Curk, T., Kayikci, M., Zupan, B., et al. iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J. Vis. Exp. (50), e2638, doi:10.3791/2638 (2011).

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Abstract

RNA结合蛋白的独特的成分和空间布局上的谈话全文(限制性商业惯例)的不同方面的指导后转录调控 1 。因此,对在分子水平上理解转录调控的一个重要步骤是获得关于限制性商业惯例 2的结合位点的位置信息。

研究蛋白质- RNA的相互作用可以使用生化方法,但这些方法没有解决的RNA在其本土蜂窝范围内具有约束力。研究蛋白- RNA复合物在细胞环境中的初步尝试,采用差异显示或微阵列分析(RIP - CHIP)3-5相结合的亲和纯化或免疫。这些方法很容易识别或间接非生理的相互作用 6 。为了增加特异性和位置的决议,这一战略被称为剪辑(紫外光交联和免疫)7,8。剪辑结合紫外光交联蛋白质和核糖核酸分子,与严格的净化计划,包括变性聚丙烯酰胺凝胶电泳。高通量测序技术相结合,作为一个强大的工具来研究蛋白质- RNA的相互作用,在全基因组规模(简称HITS - CLIP或剪辑,SEQ)9,10剪辑已经证明。近日,PAR - CLIP介绍,使用交联11,12光反应的核苷类似物。

尽管所获得的数据的高特异性,剪辑实验往往产生cDNA文库的有限序列的复杂性。这部分是由于合作纯化的RNA的限制数量和两个低效RNA结扎库准备所需的反应。此外,引物延伸分析表明,许多的cDNA核苷酸交联13提前截断。这种截断cDNA的是失去了在标准的剪辑库准备协议。我们最近开发iCLIP(单个核苷酸分辨率的),它可以捕获截断的cDNA取代低效分子RNA结扎的步骤之一,更有效的分子内的基因函证图1 )14。更重要的是,被截断的cDNA测序提供了交叉链接网站的核苷酸决议的立场的见解。我们成功地应用于iCLIP研究核蛋白C的全基因组规模的粒子组织和评估拼接的 14作用。

Protocol

1。 UV交联的组织培养细胞

  1. 取出介质,并加入6毫升冰冷的PBS细胞生长在一个10厘米的板(足够的三个实验)。
  2. 取下盖板,将在冰上。 150兆焦耳/厘米2在254 nm,照射一次。
  3. 收获的细胞与细胞起苗刮。
  4. 将2毫升细胞悬液,每3微管。最高时速为10秒旋转4 ° C至沉淀细胞,然后去除上清液。
  5. 管理单元冻结在-80 ° C,直到使用干冰和存储单元颗粒。

2。珠准备

  1. 加入100μL蛋白一个磁珠每到一个新的微管实验(DYNAL 100.02)(使用鼠标或山羊抗体蛋白G磁珠)。
  2. 洗珠裂解液(50毫米的Tris - HCl,pH值7.4; 100毫米氯化钠,1%NP - 40,0.1%SDS,0.5%去氧胆酸钠,1 / 100的蛋白酶抑制剂的鸡尾酒第三,Calbiochem)的2倍。
  3. 在2-10微克抗体100μL裂解液重悬珠。
  4. 在室温为30-60分钟旋转管。
  5. 900μL裂解液洗3倍,并准备继续执行步骤4.1离开,直到在最后一次洗涤。

3。细胞裂解和RNA的消化部分

  1. 1毫升裂解液转移到1.5 ml的微管,重悬细胞沉淀。
  2. 我核糖核酸(Ambion公司,AM2295)准备一个1 / 500稀释。添加10μL核糖核酸我稀释以及2μL涡轮的DNA酶的细胞裂解液(核糖核酸我1 / 500稀释低RNase的1 / 50稀释用于库准备; [高核糖核酸酶抗体的特异性是必要的控制) 。
  3. 孵育整整3分钟,在37 ° C在1100 rpm时的晃动,样品。立即传送到冰。
  4. 旋转4 ° C和20分钟的22000克,以明确的裂解。仔细收集上清(留约50μL裂解液沉淀)。

4。免疫沉淀

  1. 删除从珠(步骤2.5)缓冲液洗,再加入细胞裂解液(步骤3.4)。
  2. 旋转2 h后的样品在4 ° C。
  3. 弃上清,洗珠900μL高盐缓冲液(50毫米的Tris -盐酸,pH值7.4; 1 M氯化钠; 1毫米EDTA,1%NP - 40,0.1%SDS,0.5%去氧胆酸钠)2X。
  4. 洗2倍,而与900μL洗涤缓冲液(20毫米的Tris - HCl,pH值7.4; 10毫米MgCl 2的 0.2%Tween - 20的)。

5。去磷酸化的RNA的3'ends

  1. 弃上清,重悬在20μLPNK混合珠(15μl水; 4μL5倍PNK pH值6.5缓冲液[350mMTris盐酸,pH值6.5; 50mMMgCl 2 25mMdithiothreitol]; 0.5μLPNK酶0.5μLRNasin [Promega公司]) 。
  2. 20分钟在37 ° C。
  3. 加入500μl洗涤缓冲液,并用高盐缓冲液1X。
  4. 洗涤缓冲液洗2倍。

6。链接器结扎RNA 3'末端

  1. 小心取出上清液,重悬在20μL结扎混合珠(9μl水; 4μL4X连接缓冲液[200 mMTris -盐酸;40米MM GCL 2; 40毫米二硫苏糖醇]; 1μLRNA连接酶0.5μL,RNasin [NEB]; Promega公司] 1.5μL预adenylated连接器L3 [20微米; 4μLPEG400 [81170,Sigma公司)。
  2. 过夜孵育在16 ° C。
  3. 加入500μl洗涤缓冲液,然后用1毫升高盐缓冲的2倍。
  4. 洗用1 ml缓冲液洗2倍和1毫升洗第二次离开。

7。 RNA的5'末端标记

  1. 取出上清液,重悬在8μL热PNK混合珠(0.4μLPNK [NEB]; 0.8μL32个P -γ- ATP的0.8μL10X PNK缓冲[NEB]; 6μl水)。
  2. 5分钟在37 ° C。
  3. 取出热PNK组合和20μL1X Nupage样缓冲液(Invitrogen公司)重悬珠。
  4. 在thermomixer孵育70 ° C时为10分钟。
  5. 立即将一块磁铁沉淀空珠和负载上清液凝胶(见第8步)。

8。 SDS - PAGE和膜内转印

  1. 负载的样品在4-12%NuPAGE双三凝胶(Invitrogen公司)根据制造商的指示。使用1X MOPS运行缓冲液(Invitrogen)的0.5升。还可以加载5μL预染蛋白大小的标记(例如页统治者加,Fermentas公司,SM1811)。
  2. 运行50分钟的凝胶在180 V。
  3. 取出凝胶前和丢弃的固体废物(含免费放射性ATP的)。
  4. 蛋白- RNA复合物,从凝胶转移到硝酸纤维素膜,使用NOVEX调湿装置,根据制造商的说明(Invitrogen公司,转让1小时在30 V)。
  5. 转让后,PBS缓冲液冲洗膜,然后在保鲜膜包装,并到富士胶片暴露于-80 ° C(地方一个荧光贴纸膜后ALIGN T他的膜和膜30分钟,1小时,在夜间执行曝光)。

9。 RNA分离

  1. 从低核糖核酸作为面膜的步骤8.5中使用的放射自显影的实验分离蛋白- RNA复合物。这片膜切成若干小片,放入1.5 ml的微管。
  2. 添加200μLPK缓冲区(100毫米的Tris - HCl pH 7.4的50 mM氯化钠10毫米EDTA)和10μL蛋白酶K(罗氏,03115828001)膜件。孵育20分钟1100转的摇晃,在37 ° C。
  3. PKurea缓冲区(100毫米的Tris - HCl pH 7.4的50 mM氯化钠10毫米EDTA; 7 M尿素)加入200μL和20分钟在37 ° C。
  4. 收集解决方案,并添加400μLRNA的酚/氯仿(Ambion公司,9722)2毫升锁相凝胶重型管(713-2536,VWR)。
  5. 孵育5分钟在30 ° C,在1100 rpm时的晃动。 13000 RPM在室温5分钟旋转分离的阶段。
  6. 转移到一个新的管水层(要小心,不要触摸移液器凝胶)。添加0.5μLglycoblue(Ambion公司,9510)和40μL3 M醋酸钠pH值5.5和混合。然后加入1 ml 100%的乙醇,再次组合,在夜间沉淀于-20 ° C

10。反转录

  1. 20分钟转速15,000 RPM和4 ° C。去除上清液,并用0.5毫升80%乙醇沉淀。
  2. 7.25μLRNA /引物组合(6.25μl水0.5μLRclip底漆[0.5pmol/μl]; 0.5μLdNTP混合液[10毫米),重悬沉淀。对于每个实验或复制,使用不同Rclip的引物,包含个人的条码序列(见14)。
  3. 孵育5分钟,在70℃前冷却至25 ° C。
  4. 添加2.75μLRT组合(2μL的5倍RT缓冲; 0.5μL0.1M数码地面电视服务; 0.25μL标III逆转录Invitrogen公司])。
  5. 孵育5分钟在25℃,20分钟,42℃,40分钟,在50 ° C和5分钟,在80℃前冷却至4 ° C。
  6. 加入90μLTE缓冲液,0.5μLglycoblue,10μL醋酸钠pH值5.5和混合。然后加入250μL100%的乙醇,混合和过夜沉淀于-20 ° C

11。凝胶纯化的cDNA

  1. 降速和洗的样本(见10.1),然后重新悬浮颗粒在水6μL。
  2. 加入6μL的2倍TBE -尿素样缓冲液(Invitrogen公司)。加热样品至80 ° C为直接装载前3分钟。
  3. 装入预制6%TBE -尿素凝胶(Invitrogen)的样品和制造商的180 V 40分钟运行。此外,负载低分子量标记,为后续的切削(见下文)。
  4. 削减120-200新台币(高),85-120 NT(中)和70-85 NT(低)三个波段。使用theupper染料,塑料凝胶支持的标志,引导切除术(见图3)。请注意,Rclip底漆和三层序列合计占52 NT的剪辑顺序。
  5. 加入400μLTE和粉碎成小块,用1毫升注射器的柱塞的凝胶片。孵育2小时在1100 RPM晃动在37 ° C。
  6. 将两个1厘米的玻璃预过滤器进入一个中光学SpinX列(康宁公司,8161)(滤纸,1823010)。液体样品的部分转移到列。自旋为1分钟到1.5 ml管13000 RPM。
  7. 添加0.5μLglycoblue和40μL醋酸钠的pH值5.5,然后混合样本。添加1毫升100%的乙醇,重新组合和过夜沉淀在-20 ° C。

12。 cDNA的5'端引物的结扎

  1. 降速和洗的样本(见10.1),然后重新悬浮颗粒在8μL结扎组合(6.5μl水,0.8μL10倍CircLigase缓冲区第二; 0.4μL50毫米MnCl 2; 0.3μL; Circligase II [震中]),并培育在60 ° C 1小时
  2. 加入30μl的寡核苷酸退火组合(26μl水; 3μLFastDigest缓冲[Fermentas公司; 1μLcut_oligo [10μM])。孵育1分钟,在95 ° C。然后下降的温度每20秒1 ° C到25 ° C是达到了。
  3. 2μLBamHI位(快速Fermentas公司)在37和30分钟的孵化° C。
  4. 加入50μLTE和0.5μLglycoblue和混合。添加10μL醋酸钠pH值5.5和混合,再加入250μL100%的乙醇。再次在夜间混合和沉淀于-20 ° C

13。 PCR扩增

  1. 降速和洗的样本(见10.1),然后在19μl水重悬沉淀。
  2. 准备PCR混合物(19μL的cDNA; 1μL引物组合P5/P3公司Solexa,每10μM; 20μLAccuprime Supermix 1酶[Invitrogen公司])。
  3. 运行下面的PCR程序:94℃15秒,65℃30秒,30秒,68 ° C] 25-35个循环,68 ° C为3分,4个94 ° C,2分钟, ° C直到永远。
  4. 混合8μLPCR产物与2μL预制6%TBE凝胶上装载的5倍TBE缓冲和负载(Invitrogen)。 Sybrgreen我(Invitrogen)的凝胶染色,用凝胶成像仪分析。
  5. 在Rclip引物的条形码允许复用不同的样品的高通量测序之前提交。提交15μL测序库和存储休息。

14。链接程序和引物序列

前adenylated 3'连接器的DNA:

我们为了从IDT的DNA适配器,然后分装的20μM。]

的DNA

15。代表性的成果:

测序iCLIP库之前,实验的成功,可以监视两个步骤:膜内转印后的蛋白质- RNA复合物(步骤8.5)和PCR产物的凝胶图像(13.4步)放射自显影。在低核糖核酸样本的放射自显影,弥漫性放射性应被视为上述分子量的蛋白质(图2,样品4)。对于高核糖核酸样本,这种放射性是接近的蛋白质的分子量(图2,样品3)集中。如果没有抗体是免疫,应检测无信号(图2,样品1和2)。特异性免疫的进一步重要的控制,要么省略紫外线照射,或使用不表达的细胞蛋白质利息14。

的PCR产物(13.4步)的凝胶图像显示大小的范围内,对应纯化步骤11.4(图4,泳道4-6)cDNA的部分(高,中,低)。请注意,PCR引物P3Solexa和P5Solexa,引入一个额外的76 NT的cDNA的大小。如果没有抗体的免疫过程中使用,没有相应的PCR产物(图4,泳道1-3)应检测。引物二聚体的产品可以出现在约140 NT。

对于代表的高通量的测序和随后生物信息学分析结果看到14。

图1
图1。iCLIP协议的示意图。蛋白质- RNA复合物共价联用紫外线照射(步骤1)体内。感兴趣的蛋白质纯化与结合的RNA(步骤2-5)。为了让序列特异性逆转录吸,一种RNA适配器是RNA的3'端结扎,而放射性标记的5'端(步骤6和7)。交联蛋白- RNA复合物是免费的RNA纯化,用SDS - PAGE和膜内转印(步骤8)。 RNA是从膜回收,通过消化的蛋白质与蛋白酶K留在交叉链接核苷酸(步骤9)多肽其余。反转录(RT)在余下的多肽截断,并介绍了两种裂解适配器地区和条形码序列(10步)。大小选择删除前函证免费RT引物。下面的线性生成合适的模板进行PCR扩增(步骤11-15)。最后,高通量测序生成读取条形码序列会立即被最后的cDNA核苷酸(16步)。由于这核苷酸位于一个位置的交联核苷酸上游,结合部位,可以推导出高分辨率。

图2
图2交联的核蛋白的C - RNA复合物的放射自显影使用变性凝胶电泳和膜转移。 (α核蛋白核蛋白的C - RNA复合物从细胞提取核蛋白彗星对使用一种抗体免疫纯化,样品3和4)。 RNA部分被消化,使用低(+)或高(+)浓度的RNase。可以观察到向上移位大小的蛋白质(40 kDa)的配合物(样品4)。这种转变是不太明显,当高浓度的RNase(样品3)。放射性信号消失时没有抗体用于免疫沉淀(样品1和2)。

图3
图3。示意图6%TBE -尿素凝胶(Invitrogen公司),以指导切除iCLIP cDNA产物。凝胶是运行在180 V,导致重复的迁移模式的cDNA和染料在凝胶(光与暗蓝色)为40分钟。使用刀片切割(红线)高(H),中(M),低(L)cDNA的分数。开始在中间的淡蓝色染料,并立即上述商标切割塑料胶盒。划分中,低分数和修剪约1厘米以上的淡蓝色染料的高分数。使用的口袋和染料的指导下的垂直切割,分开不同的车道(在这个例子1-4)。标记巷(M)可以染色,成像控制后切割的尺寸。片段大小显示在右侧。

图4
图4。iCLIP使用凝胶电泳PCR扩增cDNA文库的分析。 RNA收回膜(图1)反转录和大小纯化变性凝胶电泳(图2)。三个大小比例的cDNA(高[H +]:[M]:85-120 NT和低[左]:70-85 NT),中型120-200 NT被收回,发送,重新线性和PCR扩增。输入分数的大小不同的结果可以观察到不同的粒度分布的PCR产物。由于PCR引物的cDNA引入76 NT,尺寸范围应161-196新台币为高,中等和146-161粒级低的NT 196-276台币之间。 PCR产物均缺席时没有抗体是免疫(1-3车道)。

Discussion

,由于iCLIP协议中含有各种各样的酶反应和纯化步骤,它并不总是很容易识别一个实验失败时的问题。为了控制一个或多个阴性对照确定RNA交叉链接网站的特殊性,应保持完整的实验和其后的计算分析。这些控件可无抗体的样品,非交联的细胞,或从基因敲除的细胞或组织免疫。理想的情况下,这些控制实验不应净化任何蛋白- RNA复合物,并因此应该没有信号,SDS - PAGE凝胶和PCR扩增后没有检测到产品。这些控制库的高通量测序应该返回很少有独特的序列。击倒细胞不建议作为时序控制,因为由此产生的序列仍然符合相同的蛋白质,这是小批量击倒细胞纯化的交叉链接网站。

注意事项也应考虑,以避免与以前的实验PCR产物的污染。最大限度地减少这种问题的最好办法是在空间上分开前和后的PCR步骤。理想的情况下,PCR产物和所有后续步骤的分析,应在一个单独的房间内进行。此外,实验室中的每个成员应该使用他们自己的缓冲区和其他试剂。这样可以更容易识别,污染源。

Disclosures

没有利益冲突的声明。

Acknowledgements

作者感谢ULE,勒斯科姆和祖潘实验室的所有成员,讨论和实验援助。我们感谢詹姆斯哈德菲尔德和尼克马修斯高通量测序。我们想指出,iCLIP方法这里描述的几个步骤股份与原始剪辑协议,柯克詹森和居开发实验室的罗伯特达内尔。这项工作是支持由欧洲研究理事会授予206726剪辑柱和一个长期的人类前沿科学计划奖学金JK

Materials

Name Company Catalog Number Comments
For gel electrophoresis and membrane transfer we recommend t he use of XCell SureLock® Mini-Cell and XCell IIâ Blot Module Kit CE Mark (Invitrogen, EI0002), which is compatible with the use of the different precast minigels that are specified throughout the protocol. The brand and order number of all materials used is mentioned during the protocol. The list of enzymes used in the protocol is shown in the table below.
Protein A Dynabeads Invitrogen 10001D use protein G for mouse or goat antibody
RNase I Ambion AM2295 activity can change from batch to batch
T4 RNA ligase I New England Biolabs M0204S
PNK New England Biolabs M0201S
proteinase K Roche Group 03115828001
Superscript III reverse transcriptase Invitrogen 18080044
Circligase II Epicentre Biotechnologies CL9021K
FastDigest® BamHI Fermentas FD0054
AccuPrime™ SuperMix I Invitrogen 12342010 this PCR mix gives the best results in our hands

DOWNLOAD MATERIALS LIST

References

  1. Keene, J. D. RNA regulons: coordination of post-transcriptional events. Nat Rev Genet. 8, 533-543 (2007).
  2. Wang, Z., Burge, C. B. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA. 14, 802-813 (2008).
  3. Trifillis, P., Day, N., Kiledjian, M. Finding the right RNA: identification of cellular mRNA substrates for RNA-binding proteins. RNA. 5, 1071-1082 (1999).
  4. Brooks, S. A., Rigby, W. F. Characterization of the mRNA ligands bound by the RNA binding protein hnRNP A2 utilizing a novel in vivo technique. Nucleic Acids Res. 28, E49-E49 (2000).
  5. Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays. Proc Natl Acad Sci. 97, 14085-14090 (2000).
  6. Mili, S., Steitz, J. A. Evidence for reassociation of RNA-binding proteins after cell lysis: implications for the interpretation of immunoprecipitation analyses. RNA. 10, 1692-1694 (2004).
  7. Ule, J. CLIP identifies Nova-regulated RNA networks in the brain. Science. 302, 1212-1215 (2003).
  8. Ule, J., Jensen, K., Mele, A., Darnell, R. B. CLIP: A method for identifying protein-RNA interaction sites in living cells. Methods. 37, 376-386 (2005).
  9. Licatalosi, D. D. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature. 456, 464-469 (2008).
  10. Yeo, G. W. An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Nat Struct Mol Biol. 16, 130-137 (2009).
  11. Urlaub, H., Hartmuth, K., Lührmann, R. A two-tracked approach to analyze RNA-protein crosslinking sites in native, nonlabeled small nuclear ribonucleoprotein particles. Methods. 26, 170-181 (2002).
  12. König, J. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 17, 909-915 (2010).

Erratum

Formal Correction: Erratum: iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution
Posted by JoVE Editors on 07/14/2011. Citeable Link.

A correction was made to iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. There was an error in part 2 of step 3. One of the characters had the incorrect symbol and was corrected to:

"...as well as 2 μl Turbo DNase..."

instead of:

"...as well as 2 ml Turbo DNase..."

Comments

71 Comments

  1. Hi,

    First I would like to say this latest method is really neat. I also like Julian's comment at the end of the video when he said with a big smirk, "You have to perform each of the 64 steps with 100% accuracy". :D That is epic.

    On a more serious note, I am just wondering if anyone can suggest what sort of primer I should use if I want to start by cloning my insert into TOPO vector instead of doing nextGen sequencing. Any help is appreciated.

    Paul

    Reply
    Posted by: Anonymous
    June 9, 2011 - 3:47 AM
  2. Hi Paul, thanks for your fun comment! TOPO cloning dŒsn²17;t require any specific primer, so you could use the one described in the protocol. Unless you wish to do something specific, such as concatemerization of sequences before inserting them into vector. Feel free to post more questions! Jernej

    Reply
    Posted by: Anonymous
    June 11, 2011 - 4:19 PM
  3. For more iCLIP questions and answers, use the following Googledoc: http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    June 13, 2011 - 11:28 AM
  4. Hi Jernej,
    Is it possible to use a 3' linker with a phosphorylated 5' end instead of a pre-adenylated 5' end and adding some ATP during the 3' linker ligation step? Thanks. Paul

    Reply
    Posted by: Anonymous
    June 13, 2011 - 10:13 PM
  5. Yes, just follow the protocol as described in Konig et al, NSMB ²010 (PMID ²0601959). More on Googledoc.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:48 AM
  6. Hi Jernej,

    Sorry to keep bombarding you with questions. In the supplementary section of your NSMB ²010 paper, shrimp alkaline phosphatase was used to desphosphorylate 3' ends. My understanding is that SAP can only desphosphorylate 5' ends. I am wondering if you had dephosphorylated 3' ends step using PNK before using SAP to dephosphorylate 5' ends.

    Quote from Konig et al, NSMB ²010: "For dephosphorylation of 3²4²; ends, Dynabeads were resuspended in ² µl 10&#²15; Shrimp alkaline phosphatase buffer (Promega), 17.5 µl H²O and 0.1 µl Shrimp alkaline
    phosphatase (Promega) and incubated at 37°C for 10 min with intermittent shaking (10 sec at 700 rpm followed by ²0 sec pause)."

    Thank you again for your help.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 4:04 AM
  7. We did use SAP in the NSMB protocol - it dŒsn't work as well as PNK on the 3' ends. We couldn't use PNK at the time, because PNK carryover into ligation reaction would create problems in the presence of ATP. In jove protocol, ligation reaction lacks ATP, therefore we can use PNK to dephosphorylate the 3' ends.

    Reply
    Posted by: Anonymous
    October 5, 2011 - 6:30 PM
  8. Hi Jernej,

    On 3.² it says add ²ml Turbo DNAse into the 1.5 ml tube. I am wondering if that amount is correct.

    Reply
    Posted by: Anonymous
    July 9, 2011 - 11:45 PM
  9. Hello Paul,
    you are right, it should be two micro liters. Sorry for that, I will try to have it changed,
    Julian

    Reply
    Posted by: Anonymous
    July 10, 2011 - 6:56 AM
  10. Hi Jernej, great protocol! Just a precision, the L3 oligo is a pre-adenylated DNA or RNA oligo? Not clear as the original Clip and iClip uses RNA...

    Thanks a bunch,

    Marco

    Reply
    Posted by: Anonymous
    September 12, 2011 - 3:58 PM
  11. Hi Marco. It's a DNA oligo. Best, Jernej

    Reply
    Posted by: Anonymous
    September 12, 2011 - 4:02 PM
  12. Hi Jernej,

    I am wondering if the 3²P-ATP batch that you normally use in your lab for step 6.1 always has close to a 100% reported radioactivity. What is the lowest percentage of remaining 3²P that you can usually still get away with? I can still get some decent signal when using 3²P-ATP that has ~50-60% remaining radioactivity but my bands on the films are not as intense as the one that I see in your publications. I am trying to work out the best schedule for ordering some 3²P-ATP and starting my experiments. Thanks again.

    Paul

    Reply
    Posted by: Anonymous
    September 14, 2011 - 11:32 PM
  13. We don't use ATP if it's more than two weeks old, thus we have >50% radioactivity. But signal intensity also depends on the efficiency of crosslinking and IP,and amount of protein expression in the cells.

    Reply
    Posted by: Anonymous
    September 15, 2011 - 4:23 AM
  14. Hi Jernej,
    Thank you for the protocol. What results if I reduce the cell samples to 100-1000 (not 10*6-7 cells) ? Thanks for your reply.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 12:24 AM
  15. That would be challenging. If you have an abundant protein that cross-links well to RNA, then it might be possible. So try running the radioactive protein-RNA complex on the gel - if you good signal after overnight exposure, then it's doable.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 4:52 AM
  16. Hi, Jernej !
    Thank you for the reply. I have another questions: How stable if the RNA-RNA and RNA-Protein photocrosslinking? How to degrade these proteins or remove the photocrosslinking? Thank you a lots.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 6:36 AM
  17. Hi Jernej,

    I again have some more questions. Do you still expose the nitrocellulose membrane at -80C when using phosphoimager instead of a film? I'm also wondering what exposure time your lab uses when using a phosphorimager screen.

    Secondly, I am wondering how many libraries containing different barcodes you can run together in a single flow cells.

    Thank you again Jernej. This protocol has been extremely useful.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:49 PM
  18. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  19. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  20. Cross-linking forms a covalent bond, so is irreversible (read the paper!). -80 would ruin the phosphorimager screen, so don't do it! We normally multiplex ±10 libraries.

    Reply
    Posted by: Anonymous
    November 15, 2011 - 7:53 PM
  21. Hi Jernej,

    In regards to one of the FAQs from Google docs.

    - When analysing PCR products, I see a band corresponding to the size of primer dimers, especially in the sample that was cut low from cDNA gel.

    Yes, it is common to see this band in the sample that was cut low from cDNA gel, and sometimes also in other samples. This is due to contamination from short cDNAs that only contain the sequence of RT primer. If this primer dimer is the dominant product on gel, we advise against sequencing the corresponding sample.

    I seem to be getting this short cDNA contamination all the time. Do you have any advice on how I could try to minimise the contamination? Have you ever isolated fragments of correct-size cDNA from a TBE-urea gel and sent only the isolated fragment for sequencing when you have short cDNA contaminations? Do you think that will work? I think that the concentration of L3 linker that I had used might have been too much. Thank you.

    Reply
    Posted by: Anonymous
    November 22, 2011 - 7:45 PM
  22. There are several possible reasons for this. Maybe one aspect of the protocol is not working, and therefore you are not producing any specific cDNA. If you have no cDNA input, then with enough cycles, you can amplify the primer-primer from any part of the gel. If you are using mammalian cells, try to get the protocol working first with hnRNP C or TIA with Santa cruz antibodies that we used in recent publications. Otherwise, using too much L3 can be a problem.

    Reply
    Posted by: Anonymous
    November 23, 2011 - 4:35 AM
  23. Very useful protocol. I have two questions.

    1. For dephosphorylation of RNA 3'ends, pH 6.5 PNK buffer is used, rather than the pH 7.6 buffer, provided by NEB. Have you compared these two conditions internally?
    ². In the protocol, the final PCR product is not isolated and quantitated before submitting for the sequencing. Are there any potential problems of doing these two steps? Can I isolate the PCR product and re-PCR using the same primers to get more product (for Illumina Hiseq)? Thank you.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 3:40 PM
  24. You can find more related answers in Googledoc http://goo.gl/4tSci, but short answers are also below:

    1. We haven²17;t compared conditions, but increased phophatase activity of PNK at lower pH has been reported in literature, you can read more in the Pubmed ID 1184²1²0.

    ². The PCR product needs to be quantified. We use both qPCR and bioanalyser. Normally, the products of the first PCR should look clean on the gel, otherwise it is a sign of a library that is of low complexity, and is unlikely to generate informative data. Therefore we advise against re-PCR, but it can be done as the last resource.

    Reply
    Posted by: Anonymous
    January 5, 2012 - 4:56 PM
  25. Hi Jernej,

    I noticed you use +/- 10 multiplexed libraries; I was wondering if you knew how many are necessary for a successful run (i.e. to provide sufficient distribution for cluster identification)?

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:20 PM
  26. The way the primers are designed here, no multiplexing is necessary, because the first three nucleotides in the primer sequence are random (part of randomer = NNN).

    Reply
    Posted by: Anonymous
    March 29, 2012 - 2:28 PM
  27. I appreciate your experiment. I have some qeustions.

    In this protocol, what dŒs barcode do high-throughout squencing?

    I don't understand function of barcode



    Reply
    Posted by: seung kuk P.
    May 23, 2012 - 6:45 AM
  28. Hi,
    this might be a really naive question but I'm wondering at the UV cross linking step, when you say you irradiate once, dŒ's this mean 1 min?

    Thank you!
    Zsofi

    Reply
    Posted by: Zsofia I.
    June 18, 2012 - 1:19 PM
  29. Hello, Thank you for this helpful technique, I just have a question. My experiments protocols are: 1. UV-crosslink RNA-protein; ². Isolate the RNA-protein complex by immunopricitation; 3. Isolate the binding RNA. 3²P-labeling the binding RNA. 4. Analysis the RNA by microarray.
    Because I do not need to sequence the RNA, and I only want to isolate the binding RNA for microarray analysis after UV-crosslink RNA-protein, so I wonder whether I need to do the step 5-7 in your protocols or I could skip from step 4 to step 8 in your protocol?

    Thanks very much, I look forward to your kind reply!

    Sean

    Reply
    Posted by: xiaoyun w.
    July 22, 2012 - 9:09 PM
  30. It is unlikely you will have enough cDNA for microarray hybridisation without some kind of amplification. You can try using steps 4-8, but you could also amplify in other ways.

    Reply
    Posted by: Anonymous
    July 23, 2012 - 6:03 AM
  31. Hi Jernej,

    Is there any published article on how to analyse iCLIP's high-throughput sequencing data? I have just got my sequencing results back following steps in your protocol. I want to make sure I check with you before digging into the data. Thank you.

    Reply
    Posted by: Anonymous
    August 1, 2012 - 10:15 PM
  32. The article is not yet published, but is in preparation by Tomaz Curk ( http://www.fri.uni-lj.si/en/tomaz-curk/), who made a public server: http://icount.biolab.si/. You can contact Tomaz at tomaz.curk@fri.uni-lj.si for more information.

    Reply
    Posted by: Anonymous
    August 2, 2012 - 5:47 AM
  33. Hi,
    it is so powerful technique! But I cannot IP any protein follow protocol. Is there any difference in affinity between different antibodies and their antigen? Could you give me some advice? Maybe we could decrease concentration of SDS or sodium deoxycholate?
    Thanks, I look forward to your kind reply!
    Min

    Reply
    Posted by: Min S.
    August 5, 2012 - 11:04 PM
  34. Hi Min, you can find advice on IP googledoc http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 6, 2012 - 3:35 AM
  35. Hi Jernej,

    With the barcoding system, I am just wondering if the three random nucleotides are there for indexing purpose during Illumina sequencing run but it's not necessary for splitting the different libraries later on. For RC1, the sequencing results will be something like NNNGGTTNN.... During analysis, do you usually trim the 3-bp from the 5'-end of the results and split the different replicates after the trimming step? I have just realised this was slightly different to the barcoding system used in your NSMB paper. -paul

    Reply
    Posted by: Anonymous
    August 6, 2012 - 9:41 PM
  36. Hi Paul! You can find the answer under the topic of "Use of random barcode in data analysis" in http://goo.gl/4tSci.

    Reply
    Posted by: Anonymous
    August 7, 2012 - 7:58 AM
  37. Hi Jernej,

    I started optimising CLIP couple of months ago and I'm at the stage that I'm convinced that I can efficiently cross link RNA to my protein (checked it by specific qRT PCR). I'm lucky because I don't need to fiddle with the IP since I've optimised before and works fine. But just to double check, after IP and western blotting a smear and a lower amount of original kDa protein is a good sing for cross linking yes?
    So my problems started at the RNase A step, I don't see any changes in size/appearance on WB after treatment... I'm convinced that my protein creates a massive complex (couple of 100 kDa) and it is because my target RNA is 10 kb to start with and there are at least 3 proteins binding to it. I'm working with a RNA virus, that's the explanation for it. I think the reason I don't see any change in kDA is because the complex dŒsn't even enter the gel to start up with. Although I used the given buffer which should break any membrane apart but the proteins are still there possibly protecting the RNA. Did you ever come across similar problems and would you have any suggestions? Also, I understand that the RNase trimming is necessary for the efficient RT step but is it a problem if the RNA is too long? What is too long? DŒs this depend on the RT enzyme used I recon or is this also important for the sequencing?

    I would greatly appreciate yur help because I'm stuck...

    Thank you,
    Zsofi

    Reply
    Posted by: Zsofia I.
    October 10, 2012 - 7:38 AM
  38. Hi Zsofi,

    For partial RNAse digestion we use RNase I (step 3). We use two different concentrations: a lower one that makes fragments with a mean between 50-100 bp and a higher concentration that fragments RNA to around 10bp. The lower one is used for preparing libraries, the higher one is used for analytical reasons.

    The RNAse step is important to (1) allow the protein RNA complex enter the Gel (²) to narrow down the crosslink site to a fragment with a size compatible with high throughput sequencing (maximum around 300 bp). So you definitely need to optimize this step for your experiments.

    If the complex you are studying is not covalently linked it should fall apart during the denaturing Gel run. Only a small fraction of your complex will have all the proteins of your complex crosslinked to the RNA at the same time since crosslinking is a very inefficient step. Therefore with the higher RNAse concentration you should be able to see a radioactive signal at the size of the protein you are studying.

    I hope that helps, best regards,
    Julian

    Reply
    Posted by: Julian K.
    October 11, 2012 - 9:49 AM
  39. Hi Julian,

    I have had some trouble with the RNase step when nuclease-ing the total lysate... In my troubleshooting efforts I read that RNase I is inhibited by 0.1% SDS, which is the concentration used in your lysis buffer. It dŒsn't seem that you guys have any problem though...do you think this is due to using an excess of RNase I or what? Just curiously confused. Thanks,

    sam

    Reply
    Posted by: Sam F.
    February 5, 2013 - 6:00 PM
  40. Hi Sam,

    in our experience the inhibition of RNase I by SDS is not an issue. You just optimize the concentration of RNase I to obtain the desired fragmentation. If you have problems doing that with your buffer conditions, you could also do the RNase digestion on the beads instead of in the lysate.

    Best,
    Julian

    Reply
    Posted by: Julian K.
    February 6, 2013 - 6:19 AM
  41. Thanks for the quick reply Julian. Your recommendation to do the "on bead" digestion is exactly what I have done and it seems to be working fine. Cheers

    Posted by: Sam F.
    February 6, 2013 - 10:12 AM
  42. Hi,

    I was wondering how many minutes have you irradiated the cells in case of HNRNP C?

    Reply
    Posted by: Niaz M.
    November 26, 2012 - 6:29 PM
  43. Hi Niaz,
    we are normally not measuring time of irradiation but the Energy per square centimeter:
    Step 1.²: ... Irradiate once with 150 mJ/cm² at ²54 nm.
    In our Stratalinker this takes 50s. However time of irradiation is not very informative here since it changes with the age or quality of the lamps, etc.
    Cheers, Julian

    Reply
    Posted by: Julian K.
    November 27, 2012 - 6:20 AM
  44. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  45. Hi,

    Thank you for wonderful protocol !

    I would like to confirm about adaptor and primer sequences.
    1. L3 adaptor and Rclip RT primer has ²²0;same²²1; sequences, not ²²0;complementary²²1; sequences. Are they O.K.? In my understanding, L3 and TR primers should have ²²0;complementary sequences.
    ². P3 Solexa 3²17; 11 nt sequence (TCTTCCGATCT) looks ²²0;extra²²1;. Both of P5 and P3 have the same sequence, which is complementary to Rclip RT primer or L3 adaptor. I think only P5 should have this sequence.

    Thank you for your help.

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 3:04 AM
  46. Hi Lisa,

    it is correct that the ends of P3 and P5 primers are the same. This is because of Illumina's primer design for their high throughput sequencing platform. When you look at the 3' end of the Rclip primers (after the Bamhi cleavage site) you can see that they are actually complementary to the 3'end of the L3 adapter.

    Cheers,
    Julian

    Reply
    Posted by: Julian K.
    December 18, 2012 - 10:05 AM
  47. I got it !!!
    Thank you :)

    Best,
    Lisa

    Reply
    Posted by: Risa K.
    December 18, 2012 - 1:11 PM
  48. Hi
    Thanks for the protocol. I have one question that has been bothering me, though. Both the RNA ligase and PNK buffers will expose the antibody column to relatively high dithiothreitol (DTT) concentrations (10 mM and 5 mM respectively). Why dŒsn't this destroy the column by reducing the disulphide bonds holding the heavy and light antibody chains together? Have you ever tried to improve the immunoprecipitation step by attempting to minimize the DTT concentration as much as possible or is this not an issue. Any assistance would be greatly appreciated. Thanks - Greg

    Reply
    Posted by: Greg C.
    February 3, 2013 - 2:13 PM
  49. Hi Greg, we haven't seen an effect of the DTT in the buffers on the IP efficiency, it seems that the concentration is not high enough to reduce the IgG - however, it is worth testing this the first time you do IP, since it is plausible that this will vary dependent on the source of your buffers (company used for PNK and ligase), or antibodies.

    Reply
    Posted by: Anonymous
    February 6, 2013 - 2:45 AM
  50. Hi Jernej,
    Thanks for the reply. The antibody I am using is definitely sensitive to the level of DTT found in the PNK buffer and I need to limit the over-all exposure of the column to DTT as much as possible. As a result, rather than using PNK as the 3' phosphatase, I would like to use an alkaline phosphatase. I noticed that in your ²010 NSMB paper you are using Shrimp Alkaline Phosphatase and in your ²009 Methods paper you use FAST AP. Did you find that the Shrimp phosphatase is significantly better ?

    Thanks again - Greg

    Reply
    Posted by: Greg C.
    February 8, 2013 - 3:52 PM
  51. Hi Greg, we don't have any evidence to suggest that one is better than the other for the on-bead reaction. At the time we were using SAP in the lab generally since it can be heat-inactivated, so therefore we also used it for on-bead (even though here you can't heat-inactivate it on beads). So you can go ahead with either one.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:29 AM
  52. I should also add that even though we didn't compare FAST AP and SAP, we did compare SAP with PNK, and we had a lot better results with PNK. It seems that SAP is not efficient as a 3' phosphatase. So it may be better for you to determine the minimal DTT amount in the buffer that is compatible with your antibody, and then continue using it with PNK and ligase. If you use fresh DTT, 1mM is likely to be sufficient both for PNK and RNA ligase.

    Reply
    Posted by: Anonymous
    February 9, 2013 - 5:39 AM
  53. Thanks, I really appreciate the advice.
    -Greg

    Posted by: Greg C.
    February 9, 2013 - 9:03 AM
  54. Hi, thanks for the awesome video. I have two questions related to the reagents:
    1. What concentration is the PEG400? (it only says 4 ul in the protocol).
    ². Under "Reverse transcription", step 6, what is the pH of the TE buffer you use? Is it pH 8?
    Thank you very much for your help. -QT

    Reply
    Posted by: Qiumin T.
    February 7, 2013 - 1:22 PM
  55. Hi QT,
    (1) we are using PEG400 from Sigma (²0²398). It is a viscous liquid.
    (²) Yes, the it is pH 8
    Cheers, Julian

    Reply
    Posted by: Julian K.
    February 7, 2013 - 4:56 PM
  56. Thank you so much Julian. I have another question. Could you recommend a protocol for doing iCLIP with mouse brain tissue? Do you know whether the tissue prep steps from this protocol ( http://ago.rockefeller.edu/Ago_HITS_CLIP_Protocol_June_²009.pdf) will work well for iCLIP as well?

    Reply
    Posted by: Anonymous
    February 13, 2013 - 5:52 PM
  57. This protocol should be fine. We also recently published a bookchapter about the iCLIP protocol which contains information on tissue samples and lots of other useful info and background:
    http://onlinelibrary.wiley.com/doi/10.100²/97835²764458².ch10/summary

    Reply
    Posted by: Julian K.
    February 14, 2013 - 5:19 AM
  58. The pre-publication version of the book chapter is available here: http://www².mrc-lmb.cam.ac.uk/groups/jule/publications/Konig_wiley.pdf.

    Reply
    Posted by: Anonymous
    February 19, 2013 - 1:54 PM
  59. I enjoyed reading about your updated iCLIP protocol in the book Tag-based Next Generation Sequencing from Wiley. I would be grateful for more details about the amount and activity of the 3²-P that you use to radioalabel the RNA. In both the book chapter and the JoVE article I see only volumes, not activities.

    In the figure for step 9, the radiolabel on the 5' end of the RNA is missing, but shouldn't it still be there? At what point in the protocol can we be reasonably sure that we are dealing with unlabeled material?

    Also, have you ever explored non-radioactive approaches to labeling, or is the sensitivity of these methods too low for the purposes of this protocol?

    Thanks!

    John

    Reply
    Posted by: John S.
    June 17, 2013 - 9:23 AM
  60. Dear John,

    with the current protocol most of the radioactivity is gone after the gel purification of the cDNA. You can increase this effect by treating the samples with RNAse after the reverse transcription (The radioactive RNA fragments then running much faster then the cDNAs in the gel). We are currently working on a protocol where we fragment the RNA by alkaline hydrolysis, which will be available soon (We want to avoid using too much RNAse at our desks).

    In addition you should always measure your samples with a Geiger counter. If your final PCRs are still hot, then you should decrease the fraction of beads that go into the labeling reaction.

    Best wishes,
    Julian

    Reply
    Posted by: Julian K.
    June 19, 2013 - 11:52 AM
  61. Hi,
    I have a question which relies on your experience with the data generated with CLIP:
    because of the UV irradiation the protein crosslinks to the RNA which even after proteinase K treatment presents an obstruction to the reverse transcriptase which therefore either skips or adds random nucleotide(s). So my question is that how long the deletions/insertions can be? Is it only one nucleotide or can also be 20?

    Thank you very much!!!

    Reply
    Posted by: Zsofia I.
    August 8, 2013 - 12:06 PM
  62. We see that >80% of cDNAs truncate at the crosslink site, and the mutations are quite rare in the remaining sequences. All we know about crosslink-induced mutations has been published here: http://www.ncbi.nlm.nih.gov/pubmed/22863408.

    Reply
    Posted by: Anonymous
    August 8, 2013 - 12:15 PM
  63. Thanks for your protocol. I have a question about the IgG background signal in the p32 labeled Western Blot. I used mouse IgG1 isotype as a control. I did not crosslink the IgG to the beads, so IgG1 stays around 50 and 25 KDa region. I do observe some radioactivity band around 50 kDa. Do you notice this in your experiments as well? Since it is close to my protein region, can you give me some suggestions to avoid this?

    Sincerely,
    Mei

    Reply
    Posted by: Xuemei Z.
    August 9, 2013 - 1:42 PM
  64. We don't get a signal in control IP. Most likely this is an RBP that non-specifically binds under your conditions. It is important to wash with high-salt buffer, and rotate the tubes for ±5min during these washes. Also, diluting the lysate before IP may help. Standard IP optimisations, basically.

    Reply
    Posted by: Anonymous
    August 9, 2013 - 2:24 PM
  65. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:29 AM
  66. Hi,
    Thank you for wonderful protocol.
    Usually how much RNA concentration one should get after Isolation from membrane? I would appreciate your reply.

    Reply
    Posted by: Bhagya B.
    August 13, 2013 - 5:57 AM
  67. Hi,
    I have 2 more questions. In this protocol you did not remove 5' phosphate of the RNA, can you still label the 5' side with P32 by PNK later? Another question, is it possible to just p32 label the RNA, cut the band, extract, degrade the protein and add 3' linker for RT later?

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:06 PM
  68. Normal PNK has phosphatase activity, so it can replace the 5' phosphate. The original CLIP protocol from Ule et al, Science 2003 added 3' linker after RNA extraction, but as explained in Ule et al, Methods 2005., the efficiency and purity of the protocol increases if linker is ligated on beads.

    Reply
    Posted by: Anonymous
    August 21, 2013 - 2:15 PM
  69. Thanks for this amazing protocol and your rapid and very helpful exchange here in this site.

    Reply
    Posted by: Xuemei Z.
    August 21, 2013 - 2:26 PM
  70. Hello, thank you for this wonderful protocol.
    I have a question:
    -I get positive Radioactive signal at the right size of positive CTRL used in this protocol in the NOT UV samples, it looks exactly as I was using high RNAse condition. why?
    I am phosphorylating the protein? is it possible?
    thank you

    Reply
    Posted by: jessica c.
    February 27, 2016 - 8:45 PM
  71. Hi Jessica, you are right, if you see signal in the non-UV control, this means that the protein is getting phosphorylated in some other way. If it has a kinase domain it may even phosphorylate itself. Or maybe some kinase is getting co-purified? You could check for this by omitting PNK from the phosphorylation reaction.

    Reply
    Posted by: Jernej U.
    March 16, 2016 - 11:07 AM

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