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[Bio101] Crosslinking and Immunoprecipitation in Zebrafish (Originally by S. Little)
[Bio101] 斑马鱼胚胎中蛋白交联和免疫沉淀(最初由S. Little 撰写)   

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Abstract

Immunoprecipitation (IP) is a routine method to detect protein binding and interactions. But the weak binding between two proteins is often hard to detect during regular IP procedure. This protocol offers a crossliking and IP combination method to detect weak binding of proteins in zebrafish embryos.

Materials and Reagents

  1. NaCl
  2. KCl
  3. CaCl2
  4. Tris
  5. EDTA
  6. Glycerol
  7. Triton X-100
  8. TBST
  9. Glycine
  10. NP-40
  11. Na deoxycholate
  12. SDS
  13. DTSSP (Thermo Fisher Scientific, catalog number: 21578 )
  14. HEPES (Life Technologies, Invitrogen™, catalog number: 15630 )
  15. Protease inhibitors (Sigma-Aldrich, catalog number: P2714 )
  16. Anti-HA affinity matrix (Roche Diagnostics, catalog number: 11815016001 )
  17. Anti-FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220 )
  18. Modified Ringer’s solution (see Recipes)
  19. Lysis buffer (see Recipes)
  20. RIPA (see Recipes)
  21. Wash buffer (see Recipes)

Equipment

  1. Kontes tubes

Procedure

  1. Protein analysis
    a.  Inject embryos with RNA encoding epitope-tagged factors at the one cell stage, manually dechorionate, and allow to develop until early gastrulation (shield stage, 6 hpf).
    b.  Determine amount of RNA to be injected by the amount needed to rescue respective mutants or morphants.
    Note: The RNA amount determines the number of embryos processed for each IP, with more embryos required in cases where relatively less RNA was injected per embryo.
  2. Crosslinking of receptors
    c.  Place embryos at shield stage in 0.2 ml modified Ringer’s solution containing 5 mM DTSSP.
    d.  Incubate embryos at 28 °C for 1.5 h, then transfer into Ringer’s plus 50 mM Tris pH 7.6 and incubate at room temperature for 20 min to quench the crosslinking reaction.
    Note: No crosslinking was performed for ligand IPs.
    e.  Transfer embryos into 0.2 to 0.4 ml lysis buffer, disrupt manually in Kontes tubes with pestle, and incubate on ice for 30 min with vortexing every 5 min.
    f.  Clarify by 30 min centrifugation, and transfer supernatant to fresh tubes.
  3. Immunoprecipitation (IP)
    g.  For HA epitope, use 2 μl packed resin per sample anti-HA affinity matrix added directly to embryo lysates.
    h.  For FLAG epitope, use 2.5 μl packed gel per sample anti-FLAG M2 affinity gel prepared by washing four times briefly in excess TBST, once for 10 min in 0.1 M glycine pH 3.5, four times briefly in TBST, and once in lysis buffer.
    i.  Expose samples to affinity gel overnight at 4 °C with gentle mixing.
    j.  Wash receptor IPs six times in RIPA for one hour per wash, followed by one overnight wash.
    k.  Wash ligand IPs three times briefly in wash buffer.
    l.  After washes, leave affinity resin in 10 μl buffer, then add 10 μl 2x SDS loading buffer.
    m.  Store samples at 4 °C until SDS-PAGE analysis.

Recipes

  1. Modified Ringer’s solution (pH 7.8)
    116 mM NaCl
    3 mM KCl
    4 mM CaCl2
    5 mM HEPES
  2. Lysis buffer
    50 mM Tris (pH 7.5)
    150 mM NaCl
    1 mM EDTA
    10% glycerol
    1% Triton X-100
    Protease inhibitors
  3. RIPA
    50 mM Tris (pH 8.0)
    150 mM NaCl
    1% NP-40
    0.5% deoxycholate
    0.1% SDS
    Protease inhibitors
  4. Wash buffer
    50 mM Tris (pH 7.6)
    150 mM NaCl
    1% Triton X-100
    Protease inhibitors

Acknowledgments

This protocol was adapted from Reference 1, and tested and developed in the Michael Granato Lab at University of Pennsylvania, Philadelphia, USA. This work was supported by NIH grant R01HD037975.

References

  1. Little, S. C. and Mullins, M. C. (2009). Bone morphogenetic protein heterodimers assemble heteromeric type I receptor complexes to pattern the dorsoventral axis. Nat Cell Biol 11(5): 637-643.

简介

免疫沉淀(IP)是检测蛋白质结合和相互作用的常规方法。 但两个蛋白质之间的弱绑定往往很难在常规IP过程中检测。 这个协议提供crossliking和IP组合方法来检测斑马鱼胚胎中蛋白质的弱绑定。

材料和试剂

  1. NaCl
  2. KCl
  3. CaCl 2
  4. Tris
  5. EDTA
  6. 甘油
  7. Triton X-100
  8. TBST
  9. 甘氨酸
  10. NP-40
  11. 脱氧胆酸钠
  12. SDS
  13. DTSSP(Thermo Fisher Scientific,目录号:21578)
  14. HEPES(Life Technologies,Invitrogen TM,目录号:15630)
  15. 蛋白酶抑制剂(Sigma-Aldrich,目录号:P2714)
  16. 抗HA亲和基质(Roche Diagnostics,目录号:11815016001)
  17. 抗FLAG M2亲和凝胶(Sigma-Aldrich,目录号:A2220)
  18. 改进的林格氏溶液(参见配方)
  19. 裂解缓冲液(见配方)
  20. RIPA(参见配方)
  21. 洗涤缓冲液(见配方)

设备

  1. Kontes管

程序

  1. 蛋白质分析
    a。 在一个细胞阶段注射胚胎与RNA编码表位标记的因素,手动dechorionate,并允许发展,直到早期gastrulation(屏蔽阶段,6 hpf)。
    b。 通过拯救相应突变体或morphant所需的量确定待注射的RNA的量。
    注意:RNA量决定了每个IP处理的胚胎数量,在每个胚胎注射相对较少的RNA的情况下需要更多的胚胎。
  2. 受体的交联
    c。 放置胚胎在屏蔽阶段在0.2毫升改性林格氏溶液含有5毫米的DTSSP。
    d。孵育胚胎在28℃下1.5小时,然后转移到林格氏加50mM Tris pH 7.6,并在室温下孵育20分钟猝灭交联反应。
    注意:对配体IP没有进行交联。
    e。 转移胚胎到0.2到0.4毫升裂解缓冲液,手动破碎在Kontes管用杵,并在冰上孵育30分钟,每5分钟涡旋。
    f。 通过30分钟离心澄清,并将上清液转移到新鲜管中。
  3. 免疫沉淀(IP)
    g。 对于HA表位,使用每个样品抗HA亲和基质直接添加到胚胎裂解物中的2μl填充树脂。
    h。  对于FLAG表位,使用2.5μl填充凝胶/样品抗FLAG M2亲和凝胶,通过在过量TBST中短暂洗涤四次,在0.1M甘氨酸pH 3.5中洗涤10分钟,在TBST中短暂洗涤四次,在裂解缓冲液中洗涤一次。
    i。  将样品在4℃下温和混合下暴露于亲和凝胶过夜。
    j。  在RIPA中洗涤受体IPs六次,每次洗涤一小时,然后一次过夜洗涤 k。  在洗涤缓冲液中将配体IP洗涤三次。
    l。  洗涤后,将亲和树脂置于10μl缓冲液中,然后加入10μl2×SDS上样缓冲液 m。  将样品储存在4℃直至SDS-PAGE分析

食谱

  1. 改性林格氏溶液(pH7.8)
    116 mM NaCl 3 mM KCl
    4mM CaCl 2
    5 mM HEPES
  2. 裂解缓冲液
    50mM Tris(pH7.5) 150mM NaCl 1mM EDTA
    10%甘油 1%Triton X-100 蛋白酶抑制剂
  3. RIPA
    50mM Tris(pH8.0) 150mM NaCl 1%NP-40
    0.5%脱氧胆酸盐 0.1%SDS
    蛋白酶抑制剂
  4. 洗涤缓冲液
    50mM Tris(pH7.6) 150mM NaCl 1%Triton X-100 蛋白酶抑制剂

致谢

该方案改编自参考文献1,并在美国宾夕法尼亚大学(University of Pennsylvania,Philadelphia,USA)的Michael Granato实验室中测试和开发。 这项工作是由NIH授权R01HD037975支持。

参考文献

  1. Little,S.C。和Mullins,M.C。(2009)。 骨形态发生蛋白异质二聚体组装异聚I型受体复合物以形成背轴模式。 et al。Nat Cell Biol 11(5):637-643。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2012). Crosslinking and Immunoprecipitation in Zebrafish (Originally by S. Little) . Bio-protocol Bio101: e174. DOI: 10.21769/BioProtoc.174;
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