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ImmunoPrecipitation of Nuclear Protein with Antibody Affinity Columns
采用抗体亲和色谱进行细胞核蛋白免疫沉淀试验

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Abstract

Co-Immunoprecipitation (Co-IP) is the method used to pull down protein partners of a protein of interest using an antibody that specifically binds to this specific protein in order to test protein-protein interaction. “Pulled down” proteins can be analyzed by western blot for suspected protein partner, or by Mass spectrometry for high throughput protein partner identification. The advantage of this technique is that endogenous protein partners can be identified from cell lines that naturally express these factors.
This protocol is optimized for hard-to-extract nuclear proteins, e.g., that stick to the nuclei inclusion bodies / nucleosome complexes such as TLX1 and TLX3 (Dadi et al., 2012). Most often, these factors are not soluble when using classical protein extraction methods. We used to add nucleases in order to increase solubilization of protein complexes trapped within inclusion bodies; though the efficacy varies depending on the given protein and therefore has to be empirically determined.

Keywords: Biochemistry(生物化学), Protein(蛋白), ImmunoPrecipitation(免疫沉淀)

Materials and Reagents

  1. Proteine G agarose beads (Upstate, Millipore Corporation, catalog number: 16-266 )
  2. Dimethylpimelimidate (Sigma-Aldrich)
  3. Sodium borate
  4. Merthiolate
  5. Ethanolamine
  6. Merthiolate
  7. Protease inhibitor cocktail, EDTA free (F. Hoffmann-La Roche, catalog number: 04693159001 )
  8. Benzonase nuclease (Sigma-Aldrich, catalog number: E1014 )
  9. 2-mercapthoethanol
  10. Bromophenol Blue
  11. NP40
  12. Sucrose
  13. CaCl2
  14. MgOAc
  15. EDTA
  16. DTT
  17. PMSF
  18. DNase I (Sigma-Aldrich, catalog number: DN25 )
  19. Laemmli buffer (see Recipes)
  20. Sucrose buffer (see Recipes)

Equipment

  1. Wheel in a cold room
  2. Refrigerating centrifuge 1.5 ml tubes

Procedure

  1. Antibody affinity columns
    1. Incubate 5 to 10 mg of the antibody (Ab) per 1 ml of wet washed protein G agarose beads for 1 hour at room temperature with gentle rocking. The quantity of Ab and protein G agarose beads varies and depends on the affinity of the Ab (see note 1).
    2. Wash the beads twice with 10 volumes of 0.2 M sodium borate (pH 9.0) by centrifugation at 3,000 x g for 30 sec.
    3. Resuspend the beads in 10 volumes of 0.2 M sodium borate (pH 9.0) and save 1% of the total beads volume (aliquot “before”). Add dimethylpimelimidate (solid) to a 20 mM concentration. The pH is critical and should be above 8.3 after adding the dimethylpimelimidate for efficient coupling and can be checked with pH strips indicator for example.
    4. Mix for 30 min at room temperature on a rocker or shaker. Save 1% of the total beads volume (aliquot “after”).
    5. Centrifuge at 3,000 x g for 3 min and discard the supernatant.
    6. Stop the reaction by washing the beads once with equal volume of 0.2 M ethanolamine (pH 8.0). Centrifuge at 3,000 x g for 3 min and discard the supernatant.
    7. Repeat the 0.2 M ethanolamine wash one more time and incubate for 2 h at room temperature in with gentle mixing.
    8. Wash beads with equal volume of PBS. Centrifuge at 3,000 x g for 3 min and discard the supernatant. Repeat the wash with PBS one more time, then store the beads in PBS with 0.01% merthiolate in the desired volume. The beads are stable for over 1 year if stored at 4 °C.
    9. Check the efficiency of coupling by boiling samples of beads taken before and after coupling in Laemmli buffer. Run on 10% SDS PAGE and stain with Coomassie blue. Good coupling is indicated by heavy-chain bands (55 kDa) in the “before” but not in the “after” lanes. If there are small amounts of heavy chain, on the “after”, prewash the coupled beads with 100 mM glycine (pH 3.0) by centrifugation at 10,000 x g for 30 sec to remove any residual antidodies that are not covalently bound to the beads. Then wash beads with PBS then store the beads in PBS with 0.01% merthiolate in the desired volume.
  2. Nuclear extract preparation
    We used this protocol with ALL-SIL and DND41 cell lines derived from patient T cell lymphoblast.
    1. Wash cells with cold 1x PBS then centrifuge for 6 min at 1,200 rpm at 4 °C. From now on, all the steps should be performed on ice.
    2. Resuspend the cell pellet in the chilled Sucrose buffer (5 μl/1 x 106 cells).
    3. Add vol/vol Sucrose buffer containing 0.5% NP40 (final concentration 0.25%).
    4. Mix by pipetting on ice. Save a small aliquot (1 to 5% -aliquot 1).
    5. Centrifuge 10 min at 1,100 x g at 4 °C. The pellet contains the nuclei and looks nacreous to white. The supernatant contains the cytoplasmic protein extract and can be saved if a cytoplasmic protein is of interest for Co-IP. Save a small aliquot (1 to 5% -aliquot 2).
    6. Wash the pellet with Sucrose buffer (without NP40) and centrifuge 10 min at 2,000 rpm at 4 °C.
    7. Depending on the nuclear proteins to be purified, you can lyse nuclei with
      1. Either the Nuclear Lysis buffer for soluble proteins (5 μl/ 1 x 106 cells);
      2. Or the Nuclei Lysis buffer for hard-to-extract proteins (5 μl/ 1 x 106 cells) and add DNase 5 U/μl and Benzonase 5 U/μl. Resuspension is hard since it’s very viscous with DNA.
    8. Incubate on a wheel at 4 °C for 45 min to 1 h. Save a small aliquot (1 to 5% -aliquot 3).
    9. Centrifuge 3 min at 10,000 x g at 4 °C. The supernatant is the protein nuclei extract that will serve for the IP. Save a small aliquot from the supernatant (1 to 5% -aliquot 4; which is also the input of the IP experiment) and the pellet is the insoluble substance such as membrane debris. Resuspend the pellet in 5 μl/ 1 x 106 cells of 150 mM NaCl, 10 mM Tris. Save a small aliquot (1 to 5% -aliquot 5).
    10. Verify the efficiency of the lysis by analyzing by SDS-PAGE and western-blot the presence of your proteins of interest in the saved aliquots.
      1. Aliquot 1: total cells
      2. Aliquot 2: cytoplasm protein extract
      3. Aliquot 3: total nuclei extract
      4. Aliquot 4: nuclear protein extract
      5. Aliquot 5: insoluble nuclei extract
  1. Co-ImmunoPrecipitation
    1. Incubate the protein nuclei extract with the Antibody-bound beads. (Ab concentration needs to be determined experimentally.) Optimization is required for efficient IP of your protein of interest and the quantity of Ab may vary according to the quality and affinity of the Ab, the protein G coupling, the protein stability, etc.
    2. Incubate 2 h at 4 °C with gentle rocking.
    3. Wash 4 to 6 times the beads in 100 mM NaCl, 15 mM Tris, HCl pH 7.8, by mixing by pipetting and centrifuging 10,000 x g for 30 sec at 4 °C.
    4. Elute the bound proteins in Laemmli loading buffer and separate by SDS-PAGE and analyze by western blot.

Recipes

  1. Laemmli buffer
    20 % Glycerol
    4% SDS
    250 mM Tris (pH 6.8) (stacking buffer for upper gel of SDS PAGE)
    1.4 M 2-mercapthoethanol, a pinch of bromophenol blue.
  2. Sucrose buffer
    0.32 M Sucrose
    3 mM CaCl2
    2 mM MgOAc
    0.1 mM EDTA
    10 mM DTT
    0.5 mM PMSF
  3. Nuclei Lysis buffer for hard to extract nuclear factors
    50 mM Hepes (pH 7.8) 
    3 mM MgCl2
    300 mM NaCl
    1 mM DTT
    0.1 mM PMSF
    Protease inhibitor complete mini EDTA free tablets 1x
  4. Nuclei Lysis buffer for soluble nuclear factors
    50 mM Hepes (pH 7.8) 
    50 mM KCl
    300 mM NaCl
    0.1 mM EDTA
    10 % Glycerol
    1 mM DTT
    0.1 mM PMSF
    Protease inhibitor complete mini EDTA free tablets 1x

Notes

  1. We used Protein G agarose beads with mouse IgG1 antibodies. However, the affinity of the isotype antibody of interest needs to be verified accordingly to the protein A or G manufacturer’s recommendations.
  2. The quantity of the cells used for protein extract may vary according to the expression, the stability of the protein.
  3. The Benzonase and DNase treatment is used only in case of hard to extract proteins that stick to the nuclei inclusion bodies/ nucleosome. If the proteins of interest are soluble, the use of the Nuclei Lysis buffer for soluble factors is preferred.

Acknowledgments

Work in the PF laboratory is supported by institutional grants from 'Institut National de la Santé et de la Recherche Médicale' (Inserm) and 'Centre National de la Recherche Scientifique' (CNRS), and by dedicated grants from the Commission of the European Communities, the 'Agence Nationale de la Recherche' (ANR), the 'Institut National du Cancer' (INCa), the 'ITMO Cancer Alliance Nationale pour les Sciences de la Vie et de la Santé' (AVIESAN) and the 'Fondation Princesse Grace de la Principauté de Monaco'. S.D. was supported by fellowships from the ‘Ministère de l’Enseignement Supérieur et de la Recherche’, the ‘Fondation pour la Recherche Médicale’ (FRM), and the ‘Société Française d’Hématologie’ (SFH).

References

  1. Dadi, S., Le Noir, S., Payet-Bornet, D., Lhermitte, L., Zacarias-Cabeza, J., Bergeron, J., Villarese, P., Vachez, E., Dik, W. A., Millien, C., Radford, I., Verhoeyen, E., Cosset, F. L., Petit, A., Ifrah, N., Dombret, H., Hermine, O., Spicuglia, S., Langerak, A. W., Macintyre, E. A., Nadel, B., Ferrier, P. and Asnafi, V. (2012). TLX homeodomain oncogenes mediate T cell maturation arrest in T-ALL via interaction with ETS1 and suppression of TCRalpha gene expression. Cancer Cell 21(4): 563-576.
  2. Dignam, J. D., Lebovitz, R. M. and Roeder, R. G. (1983). Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 11(5): 1475-1489.
  3. Gersten, D. M. and Marchalonis, J. J. (1978). A rapid, novel method for the solid-phase derivatization of IgG antibodies for immune-affinity chromatography. J Immunol Methods 24(3-4): 305-309.
  4. Schneider, C., Newman, R. A., Sutherland, D. R., Asser, U. and Greaves, M. F. (1982). A one-step purification of membrane proteins using a high efficiency immunomatrix. J Biol Chem 257(18): 10766-10769.
  5. Simanis, V. and Lane, D. P. (1985). An immunoaffinity purification procedure for SV40 large T antigen. Virology 144(1): 88-100.

简介

共免疫沉淀(Co-IP)是用于使用特异性结合该特异性蛋白的抗体来下拉目标蛋白的蛋白质配偶体以测试蛋白质 - 蛋白质相互作用的方法。 "下拉"蛋白质可通过蛋白质印迹分析用于可疑蛋白质伴侣,或通过质谱法用于高通量蛋白质伴侣鉴定。这种技术的优点是可以从天然表达这些因子的细胞系中鉴定内源性蛋白质伴侣。
该方案针对难以提取的核蛋白进行优化,例如到核内含体/核小体复合物例如TLX1和TLX3(Dadi等人,2012)。最常见的是,当使用经典的蛋白质提取方法时,这些因素是不可溶的。我们用来添加核酸酶以增加被包涵体中捕获的蛋白质复合物的溶解;尽管功效根据给定的蛋白质而变化,因此必须根据经验确定。

关键字:生物化学, 蛋白, 免疫沉淀

材料和试剂

  1. 蛋白G琼脂糖珠(Upstate,Millipore Corporation,目录号:16-266)
  2. 二甲基嘧啶亚胺(Sigma-Aldrich)
  3. 硼酸钠
  4. 鲜花
  5. 乙醇胺
  6. 鲜花
  7. 蛋白酶抑制剂混合物,无EDTA(F.Hoffmann-La Roche,目录号:04693159001)
  8. Benzonase核酸酶(Sigma-Aldrich,目录号:E1014)
  9. 2-巯基乙醇
  10. 溴酚蓝
  11. NP40
  12. 蔗糖
  13. CaCl 2
  14. MgOAc
  15. EDTA
  16. DTT
  17. PMSF
  18. DNase I(Sigma-Aldrich,目录号:DN25)
  19. Laemmli缓冲液(见Recipes)
  20. 蔗糖缓冲液(参见配方)

设备

  1. 轮椅在寒冷的房间
  2. 冷冻离心机1.5 ml管

程序

  1. 抗体亲和柱
    1. 在室温下温和摇动下,每1ml湿洗涤的蛋白G琼脂糖珠孵育5至10mg的抗体(Ab)1小时。 Ab和蛋白G琼脂糖珠的量变化并且取决于Ab的亲和力(参见注释1)。
    2. 用3,000体积的0.2M硼酸钠(pH9.0)通过在3,000×3000g离心30秒来洗涤珠子两次。
    3. 将珠子在10倍体积的0.2M硼酸钠(pH 9.0)中重悬,并保存1%的总珠体积("等于"前)。 加入二甲基庚二亚胺酯(固体)至20mM浓度。 pH是关键的,并且在加入二甲基庚二亚胺酸酯以有效偶联后应该高于8.3,并且可以例如用pH条指示剂检查。
    4. 在室温下在摇床或摇床上混合30分钟。保存总珠体积的1%(等于"之后")。
    5. 以3,000x/min离心3分钟,弃去上清液。
    6. 通过用等体积的0.2M乙醇胺(pH 8.0)洗涤珠一次来终止反应。以3,000x/min离心3分钟,弃去上清液。
    7. 重复0.2 M乙醇胺洗涤一次,并在室温下温和混合孵育2小时
    8. 用等体积的PBS洗涤珠子。以3,000x/min离心3分钟,弃去上清液。用PBS再次重复洗涤,然后将珠存储在具有所需体积的0.01%硫柳汞的PBS中。如果储存在4℃下,珠子可以稳定超过1年。
    9. 通过在Laemmli缓冲液中偶联之前和之后煮沸的珠子样品检查偶联的效率。在10%SDS PAGE上运行并用考马斯蓝染色。在"之前"但不在"之后"泳道中由重链条(55kDa)指示良好的偶联。如果存在少量重链,在"之后",通过在10,000xg离心30分钟,用100mM甘氨酸(pH 3.0)预洗涤偶联的珠子。以除去未与珠共价结合的任何残留抗体。然后用PBS洗涤珠子,然后将珠子存储在含有所需体积的0.01%硫柳汞的PBS中
  2. 核提取物准备
    我们用来自患者T细胞淋巴母细胞的ALL-SIL和DND41细胞系使用该方案
    1. 用冷的1×PBS洗涤细胞,然后在4℃下以1,200rpm离心6分钟。 从现在开始,所有的步骤应该在冰上进行。
    2. 在冷冻的蔗糖缓冲液(5μl/1×10 6个细胞)中重悬细胞沉淀。
    3. 加入含有0.5%NP40(终浓度0.25%)的vol/vol蔗糖缓冲液
    4. 通过在冰上吸移混合。 保存一小份(1至5% - 等分试样1)。
    5. 在4℃下以1,100×em离心10分钟。 颗粒含有核,看起来像白色的珍珠。 上清液含有细胞质蛋白质提取物,如果细胞质蛋白质对Co-IP感兴趣,则可以保存上清液。 保存一小份(1至5% - 等份2)。
    6. 用蔗糖缓冲液(不含NP40)洗涤沉淀,并在4℃下以2,000rpm离心10分钟。
    7. 根据要纯化的核蛋白,你可以用
      裂解细胞核
      1. 可溶性蛋白的核裂解缓冲液(5μl/1×10 6个细胞);
      2. 或用于难以提取蛋白质(5μl/1×10 6个细胞)的核裂解缓冲液,并加入DNA酶5U /μl和Benzonase 5U /μl。 重悬是很困难的,因为它与DNA非常粘。
    8. 在4℃下在轮上孵育45分钟至1小时。 保存一小份(1至5% - 等份3)。
    9. 在4℃下以10,000×g离心3分钟。 上清液是用于IP的蛋白质核提取物。 从上清液(1至5% - 等分试样4;其也是IP实验的输入)保存小等分试样,沉淀是不溶性物质例如膜碎片。 将沉淀重悬在150μlNaCl,10mM Tris的5μl/1×10 6个细胞中。 保存一小份(1至5% - 等份5)。
    10. 通过SDS-PAGE分析验证裂解效率,并通过Western-blot检测保存的等分试样中感兴趣的蛋白质的存在。
      1. 等分试样1:总细胞
      2. 等分试样2:细胞质蛋白提取物
      3. 等分试样3:总核提取物
      4. 等分试样4:核蛋白提取物
      5. 等分试样5:不溶性核提取物
  1. 免疫共沉淀
    1. 将蛋白质核提取物与抗体结合的珠孵育。 (Ab浓度需要通过实验确定。)需要对目的蛋白质的有效IP进行优化,并且Ab的量可以根据Ab的质量和亲和力,蛋白质G偶联,蛋白质稳定性等而变化。 br />
    2. 在4℃下温和摇动孵育2小时
    3. 在100mM NaCl,15mM Tris,HCl pH 7.8中洗涤4至6倍的珠子,通过吸移混合并在4℃下离心10,000×10 6秒30秒 。
    4. 洗脱在Laemmli上样缓冲液中的结合蛋白,并通过SDS-PAGE分离并通过western印迹分析

食谱

  1. Laemmli缓冲区
    20%甘油
    4%SDS
    250mM Tris(pH6.8)(SDS PAGE的上层凝胶的堆积缓冲液) 1.4 M 2-巯基乙醇,一小块溴酚蓝。
  2. 蔗糖缓冲剂
    0.32 M蔗糖
    3mM CaCl 2
    2mM MgOAc
    0.1mM EDTA
    10 mM DTT
    0.5 mM PMSF
  3. 难以提取核因子的核裂解缓冲液
    50 mM Hepes(pH 7.8)<
    3mM MgCl 2/
    300 mM NaCl
    1 mM DTT
    0.1mM PMSF
    蛋白酶抑制剂完全迷你EDTA免费药片1x
  4. 可溶性核因子的核裂解缓冲液
    50 mM Hepes(pH 7.8)<
    50 mM KCl
    300 mM NaCl
    0.1mM EDTA
    10%甘油
    1 mM DTT
    0.1mM PMSF
    蛋白酶抑制剂完全迷你EDTA免费药片1x

笔记

  1. 我们使用蛋白G琼脂糖珠与小鼠IgG1抗体。然而,所关注的同种型抗体的亲和力需要根据蛋白A或G制造商的建议来验证
  2. 用于蛋白质提取物的细胞的量可根据蛋白质的表达,稳定性而变化
  3. Benzonase和DNase处理仅在难以提取粘附于核包涵体/核小体的蛋白质的情况下使用。如果感兴趣的蛋白质是可溶的,则优选使用核裂解缓冲液用于可溶性因子

致谢

在PF实验室的工作由"国家科学技术研究所"(Inserm)和"国家研究中心"(CNRS)的机构赠款以及欧洲共同体委员会的专项赠款,"国家癌症研究所"(INCa),"ITMO癌症联盟国家癌症联盟"(AVIESAN)和"Fondation Princesse Grace"摩纳哥公国"。 S.D.得到"精神病学研究所","法国医学研究基金会"和"法国医学会学会"的奖学金的支持。

参考文献

  1. Dadi,S.,Le Noir,S.,Payet-Bornet,D.,Lhermitte,L.,Zacarias-Cabeza,J.,Bergeron,J.,Villarese,P.,Vachez,E.,Dik,WA,Millien C.,Radford,I.,Verhoeyen,E.,Cosset,FL,Petit,A.,Ifrah,N.,Dombret,H.,Hermine,O.,Spicuglia,S.,Langerak,AW,Macintyre,EA ,Nadel,B.,Ferrier,P。和Asnafi,V。(2012)。 TLX同源结构域致癌基因通过与ETS1的相互作用和TCRalpha基因的抑制来介导T-ALL中的T细胞成熟停滞表达。 Cancer Cell 21(4):563-576。
  2. Dignam,J.D.,Lebovitz,R.M.and Roeder,R.G。(1983)。 通过RNA聚合酶II在来自分离的哺乳动物细胞核的可溶性提取物中的精确转录起始。 em> Nucleic Acids Res 11(5):1475-1489。
  3. Gersten,DM和Marchalonis,JJ(1978)。固相的快速,新颖的方法用于免疫亲和色谱的IgG抗体的衍生化。免疫方法 24(3-4):305-309。
  4. Schneider,C.,Newman,R.A.,Sutherland,D.R.,Asser,U.and Greaves,M.F。(1982)。 使用高效免疫基质进行膜蛋白的一步纯化。 J Biol Chem 257(18):10766-10769。
  5. Simanis,V。和Lane,D.P。(1985)。 SV40大T抗原的免疫亲和纯化程序。 病毒学 144(1):88-100。
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引用:Dadi, S., Payet-Bornet, D. and Ferrier, P. (2013). ImmunoPrecipitation of Nuclear Protein with Antibody Affinity Columns. Bio-protocol 3(3): e319. DOI: 10.21769/BioProtoc.319.
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Samuel Wilson
Université de Sherbrooke
Hello,
I am interested in using your protocol to study a different protein. I am studying the cofactors of a transcription factor, and I would like to ascertain whether the interactions of cofactors with my protein of interest are protein-protein. I would like to treat the nuclear protein extract of a human cell line with DNase to free DNA-bound factors prior to co-immunoprecipitation, as you have done.
I have a couple of questions concerning your protocol:
(1) Is it correct that you are only using 5 μL of buffer for 1 million cells? The pellet I obtain from 1 million cells occupies more volume than than 5 μL of buffer. If this volume of buffer is correct, I am curious to know what is the advantage of using such a small volume of buffer.
(2) I purchased the DNaseI you recommend from Sigma (DN25), and I would like to know how you have dilluted this enzyme. The manufacturer suggests that the DNase has a solubility of 2 U/μL. This would mean adding a significantly larger volume of enzyme solution than was present in your initial nuclear protein extract!
Thank you for your help!
Cheers,
Samuel
3/22/2017 1:16:41 PM Reply