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Co-Streptavidin Precipitation (Co-SP) is a method to pull down protein partners of a protein of interest tagged with the streptavidin binding protein domain, and using streptavidin columns that specifically bind to Streptavidin Binding Protein (SBP) in order to test the protein-protein interactions. Proteins of interest to be tested for their interaction are artificially co-expressed in “easy to transfect” cells. Pull down proteins can be analyzed by western blot for suspected protein partner.

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Co-Streptavidin Precipitation
链霉亲和素共沉淀

生物化学 > 蛋白质 > 分离和纯化
作者: Saïda Dadi
Saïda DadiAffiliation: Centre d'Immunology de Marseille Luminy (CIML), Aix-Marseille UM2, INSERM UMR1104, CNRS UMR7280, Marseille, France
Bio-protocol author page: a215
Dominique Payet-Bornet
Dominique Payet-BornetAffiliation: Centre d'Immunology de Marseille Luminy (CIML), Aix-Marseille UM2, INSERM UMR1104, CNRS UMR7280, Marseille, France
Bio-protocol author page: a216
 and Pierre Ferrier
Pierre FerrierAffiliation: Centre d'Immunology de Marseille Luminy (CIML), Aix-Marseille UM2, INSERM UMR1104, CNRS UMR7280, Marseille, France
For correspondence: ferrier@ciml.univ-mrs.fr
Bio-protocol author page: a217
Vol 3, Iss 3, 2/5/2013, 5428 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.320

[Abstract] Co-Streptavidin Precipitation (Co-SP) is a method to pull down protein partners of a protein of interest tagged with the streptavidin binding protein domain, and using streptavidin columns that specifically bind to Streptavidin Binding Protein (SBP) in order to test the protein-protein interactions. Proteins of interest to be tested for their interaction are artificially co-expressed in “easy to transfect” cells. Pull down proteins can be analyzed by western blot for suspected protein partner.
Keywords: Biochemistry(生物化学), Protein(蛋白), Immuno-precipitation(免疫沉淀)

[Abstract] 共链霉亲和素沉淀(Co-SP)是一种拉下用链霉亲和素结合蛋白结构域标记的目的蛋白质的蛋白质配偶体,并使用特异性结合链霉亲和素结合蛋白(SBP)的链霉亲和素柱以测试蛋白质的方法 蛋白相互作用。 要测试其相互作用的感兴趣的蛋白质在"易于转染"细胞中人工共表达。 下拉蛋白可以通过蛋白质印迹分析用于可疑蛋白质伴侣。

Materials and Reagents

  1. HeLa cells (ATCC, catalog number: CCL-2 )
  2. SBP-Calmodulin Tag Expression Vector pNTAP (Inter Play N-Terminal Mammalian TAP Vector, Agilent Technologies-Stratagene, catalog number: 240101 )
  3. Lipofectamine (Life Technologies, InvitrogenTM, catalog number: 11668-027 )
  4. Fugene 6 (F. Hoffmann-La Roche, catalog number: 05061377001 )
  5. FLAG tag Expression Vector pCMV-Tag2 (Agilent Technologies-Stratagene, catalog number: 211172 )
  6. NP40
  7. Steptavidin agarose beads (Sigma-Aldrich, catalog number: S-1638 )
  8. Anti- Calmodulin antibody (Upstate, catalog number: 07-482 )
  9. Anti- Flag antibody (Sigma-Aldrich, catalog number: F1804-200UG )
  10. Protease inhibitor cocktail, EDTA free (F Hoffmann-La Roche, catalog number: 04693159001 )
  11. DNase I (Sigma-Aldrich, catalog number: DN25 )
  12. Benzonase nuclease(Sigma-Aldrich, catalog number: E1014 )
  13. Laemmli loading buffer (see Recipes)
  14. Sucrose buffer (see Recipes)
  15. Nuclei Lysis buffer for hard to extract nuclear factors (see Recipes)
  16. Nuclei Lysis buffer for soluble nuclear factors (see Recipes)

Equipment

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

Procedure

  1. Cell transfection
    1. Co-transfected 10 to 50 x 106 of HeLa cells (or other “easy to transfect cells”) with your gene of interest “1”-FLAG cloned into pCMV-Tag2 expression vector, and your gene of interest “2”-SBP-Calmodulin cloned into pNTAP, or the empty expression vector pNTAP as a control. Transfection is performed with standard transfection protocols (such as Lipofectamine, Invitrogen or Fugene 6, Roche or Calcium Chloride precipitation).

  1. Nuclear extract preparation
    24 to 48 H after transfection, the cells are lysed and nuclear extracts were prepared using the nuclear extract lysis protocol:
    1. Use trypsin to recover the cells and wash cells with cold 1x PBS then centrifuge for 6 min at 300 x g 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 Nuclei 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 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

  2. Co-Streptavidin Purification
    1. Incubate the protein nuclei extract 2 h at 4 °C with 25 μl of streptavidin agarose (bead volume). Optimization is required for efficient SP of your SBP-protein of interest and the quantity of streptavidin beads may vary according to the protein stability, etc. 
    2. Wash 4 to 6 times the beads in 100 mM NaCl, 10 mM Tris-HCl (pH 7.8), by mixing by pipetting and centrifuging 10,000 x g for 30 sec.
    3. Elute the bound proteins in Laemmli loading buffer and separate by SDS-PAGE and analyze by western blot using antibodies against Calmodulin and Flag.

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. The number of cells to be transfected need to be optimized, depending on the size and the stability of the proteins to be expressed.

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.

材料和试剂

  1. HeLa细胞(ATCC,目录号:CCL-2)
  2. SBP-钙调蛋白标签表达载体pNTAP(Inter Play N-Terminal Mammalian TAP Vector,Agilent Technologies-Stratagene,目录号:240101)
  3. Lipofectamine(Life Technologies,Invitrogen TM ,目录号:11668-027)
  4. Fugene 6(F.Hoffmann-La Roche,目录号:05061377001)
  5. FLAG标签表达载体pCMV-Tag2(Agilent Technologies-Stratagene,目录号:211172)
  6. NP40
  7. 链霉亲和素琼脂糖珠(Sigma-Aldrich,目录号:S-1638)
  8. 抗钙调素抗体(Upstate,目录号:07-482)
  9. 抗Flag抗体(Sigma-Aldrich,目录号:F1804-200UG)
  10. 蛋白酶抑制剂混合物,无EDTA(F Hoffmann-La Roche,目录号:04693159001)
  11. DNase I(Sigma-Aldrich,目录号:DN25)
  12. Benzonase核酸酶(Sigma-Aldrich,目录号:E1014)
  13. Laemmli加载缓冲液(参见配方)
  14. 蔗糖缓冲液(见配方)
  15. 用于难以提取核因子的核裂解缓冲液(参见配方)
  16. 用于可溶性核因子的核裂解缓冲液(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. 细胞转染
    1. 用感兴趣的感兴趣的基因"1"-FLAG共转染的10-50×10 6个HeLa细胞(或其他"易于转染的细胞")克隆到pCMV- Tag2表达载体和克隆到pNTAP中的感兴趣的"2"-SBP-Calmodulin基因,或空表达载体pNTAP作为对照。 使用标准转染方案(例如Lipofectamine,Invitrogen或Fugene 6,Roche或氯化钙沉淀)进行转染。

  1. 核提取物准备
    转染后24至48小时,裂解细胞,并使用核提取物裂解方案制备核提取物:
    1. 使用胰蛋白酶回收细胞和用冷的1×PBS洗涤细胞,然后在4℃以300×g离心6分钟。 从现在起,所有的步骤都应该在冰上进行
    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,您可以用以下溶解核:
      1. 可溶性蛋白质的核裂解缓冲液(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℃下离心3分钟10,000次 g 。 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

  2. 共链霉亲和素纯化
    1. 孵育蛋白质核提取2 h在4℃与25微升链霉亲和素琼脂糖(珠体积)。 需要优化是有效SP的您感兴趣的SBP蛋白,链霉亲和素珠的数量可以根据蛋白质稳定性等而变化。 
    2. 在100mM NaCl,10mM Tris-HCl(pH7.8)中洗涤4至6倍珠子,通过吸移混合并离心10,000×10 6秒30秒。
    3. 洗脱在Laemmli上样缓冲液中的结合蛋白,并通过SDS-PAGE分离,并使用针对钙调蛋白和Flag的抗体通过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 一小片溴酚蓝
  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
    50mM Hepes(pH7.8)
    3mM MgCl 2/
    300 mM NaCl
    1 mM DTT
    0.1mM PMSF
    蛋白酶抑制剂完整的迷你EDTA免费药片1x
  4. 可溶性核因子的核裂解缓冲液
    50mM Hepes(pH7.8)
    50 mM KCl
    300 mM NaCl
    0.1mM EDTA
    10%甘油
    1 mM DTT
    0.1mM PMSF
    蛋白酶抑制剂完整的迷你EDTA免费药片1x

笔记

  1. 待转染的细胞数量需要根据待表达的蛋白质的大小和稳定性进行优化。

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介导T细胞成熟停滞 在T-ALL中通过与ETS1的相互作用和TCRα基因表达的抑制。癌细胞 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,D.M。和Marchalonis,J.J。(1978)。 用于免疫亲和层析的IgG抗体的固相衍生化的快速,新颖的方法。/a> J Immunol Methods 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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How to cite this protocol: Dadi, S., Payet-Bornet, D. and Ferrier, P. (2013). Co-Streptavidin Precipitation. Bio-protocol 3(3): e320. DOI: 10.21769/BioProtoc.320; Full Text



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