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Biotinylation of Cell Surface Proteins
细胞表面蛋白的生物素化   

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Abstract

Membrane proteins are major sensors of extracellular stimuli and initiators of intracellular signal transduction, and their abundance on the cell surface in particular is often dynamically regulated even when there are no significant changes of their total abundance in a cell. This protocol is designed to biochemically label and separate membrane proteins on the plasma membrane from those in the intracellular compartments. In conjunction with co-immunoprecipitation and western blot analysis, functional analysis of dynamic interaction of membrane proteins with other membrane proteins or intracellular adaptor and effector proteins can be achieved.

Materials and Reagents

  1. HEK293 cells
  2. Poly-dL-Ornithine (PLO) (Sigma-Aldrich, catalog number: P8638 )
  3. Boric Acid (Sigma-Aldrich, catalog number: B0394 )
  4. Sulfo-NHS-SS-biotin solution (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 21331 )
  5. Neuroavidin beads (Thermo Fisher Scientific, Pierce Antibodies, catalog number: 29200 )
  6. EDTA•2H2O (MW 372.2) (Sigma-Aldrich, catalog number: E1644 )
  7. EGTA•2H2O (MW 380.4) (Sigma-Aldrich, catalog number: E4378 )
  8. NaPyrophophate (Sigma-Aldrich, catalog number: S9515 )
  9. NaF (Sigma-Aldrich, catalog number: S7920 )
  10. Na3VO4•10H2O (MW 446.1) (Sigma-Aldrich, catalog number: 5-9515 )
  11. Protease inhibitor (F. Hoffmann-La Roche, catalog number: 1873580 )
  12. NaVO3
  13. NaOH
  14. PBS
  15. CaCl2
  16. MgCl2
  17. Glycine
  18. Triton
  19. HCl
  20. 4x loading buffer
  21. Biotin quenching solution (see Recipes)
  22. PBS/CaCl2/MgCl2 (see Recipes)
  23. IP buffer (see Recipes)
  24. Stock (see Recipes)

Equipment

  1. 0.22 μm filters
  2. 12-well plates
  3. Western blot equipment

Procedure

  1. Prepare PLO solution: Add 90 mg PLO into 180 ml borate buffer (dissolve 1.668 g borate acid in 180 ml H2O and adjust pH to 8.3 using NaOH), and sterilize by filtering through 0.22 μm filters.
  2. Coat 12-well plates with PLO solution at room temperature overnight in the culture hood (1 ml per well). Wash three times with sterile water on the second day before cell plating.
  3. Plate HEK293 cells on poly-ornithine coated 12-well plates and transfect as needed.
  4. On the day of cell harvest, dissolve Sulfo-NHS-SS-Biotin in PBS/CaCl2/MgCl2 at 0.5 mg ml-1 and store on ice.
  5. Wash cells with 1 ml ice-cold PBS/CaCl2/MgCl2 twice.
  6. Biotinylation
    1. Add 0.3 ml of 0.5 mg/ml Sulfo-NHS-SS-biotin solution.
    2. Incubate on ice for 30 min, with occasional shaking.
  7. Wash three times with ice-cold quenching solution and incubate 5 min before each solution removal to allow neutralization of uncrosslinked reagents.
  8. Lyse cells with 120 μl IP buffer and incubate 10 min on ice.
  9. Transfer all contents of each well into a 1.5-ml tube and spin at Vmax (about 14,000 rpm) on a 4 °C desktop centrifuge for 10 min.
  10. Save 10 μl supernatant (added 10 μl 4x loading buffer) as offers.
    Mix 100 μl supernatants with 50 μl 50% slurry of immobilized Neutravidin and rotate for 2-3 h at 4 °C.
  11. Spin 1 min at 5,000 rpm in a 4 °C centrifuge. Remove the supernatant and wash the beads with 1 ml IP buffer by rotating the mixture for 5 min in the 4 °C room. Wash three times in total.
  12. After the final wash, remove the supernatant and add 25 μl 4x loading buffer to the beads. Mix well and put the tubes in a 65 °C water bath for 10 min to denature proteins (65 °C is chosen because we find that in boiling water, many membrane proteins form aggregates, which results in failure of proteins to migrate into acrylamide gels). Load 10 μl for western blot analysis.

Recipes

  1. Biotin quenching solution (pH 7.4)
    50 mM glycine (0.1877 g) in 50 ml PBS/CaCl2/MgCl2
  2. PBS/CaCl2/MgCl2
    PBS (+2.5 mM CaCl2, 1 mM MgCl2, pH 7.4)
  3. IP buffer (pH 7.4)
    PBS

    50 ml
    EDTA
    5 mM
    0.5 ml x 0.5 M (pH 7.4)
    EGTA
    5 mM
    0.5 ml x 0.5 M (pH 7.4)
    NaPyrophophate
    10 mM
    0.223 g
    NaF
    50 mM
    0.1 g
    NaVO3   
    1 mM   
    0.5 ml x 100 mM
    1% Triton
    1 tablet Protease inhibitor
  4. Stock
    1. 0.5 M EDTA (pH 7.4)
      93.05 g EDTA•2H2O
      500 ml ddH2O
      Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
    2. 0.5 M EGTA (pH 7.4)
      95.1 g EGTA•2H2O
      500 ml ddH2O
      Add NaOH until EDTA dissolves and add HCl to adjust pH back to 7.4.
    3. 100 mM Na3VO4
      1.84 g Na3VO4•10 H2O
      Add HCl to pH less than 10 (yellow solution). Boil the solution until clear and colorless.
    4. 10 ml aliquots and freeze at -20 °C.

Acknowledgments

This protocol was previously used in and adapted from Huang et al. (2006).

References

  1. Huang, G. N., Zeng, W., Kim, J. Y., Yuan, J. P., Han, L., Muallem, S. and Worley, P. F. (2006). STIM1 carboxyl-terminus activates native SOC, I(crac) and TRPC1 channels. Nat Cell Biol 8(9): 1003-1010.

简介

膜蛋白是胞外刺激和细胞内信号转导的启动子的主要传感器,并且它们在细胞表面上的丰度特别是经常动态调节,即使当它们在细胞中的总丰度没有显着变化时。 该协议设计为生物化学标记和分离质膜上的膜蛋白与细胞内区室中的膜蛋白。 结合共免疫沉淀和蛋白质印迹分析,可以实现膜蛋白与其他膜蛋白或细胞内接头和效应子蛋白的动态相互作用的功能分析。

材料和试剂

  1. HEK293细胞
  2. 聚dL-鸟氨酸(PLO)(Sigma-Aldrich,目录号:P8638)
  3. 硼酸(Sigma-Aldrich,目录号:B0394)
  4. 磺基-NHS-SS-生物素溶液(Thermo Fisher Scientific,Pierce Antibodies,目录号:21331)
  5. Neuroavidin珠(Thermo Fisher Scientific,Pierce Antibodies,目录号:29200)
  6. EDTA·2H 2 O(MW 372.2)(Sigma-Aldrich,目录号:E1644)
  7. EGTA·2H 2 O(MW 380.4)(Sigma-Aldrich,目录号:E4378)
  8. NaPyrophophate(Sigma-Aldrich,目录号:S9515)
  9. NaF(Sigma-Aldrich,目录号:S7920)
  10. (MW-446.1)(Sigma-Aldrich,目录号:5-9515)。< br /><
  11. 蛋白酶抑制剂(F.Hoffmann-La Roche,目录号:1873580)
  12. NaVO 3
  13. NaOH
  14. PBS
  15. CaCl <2>
  16. MgCl 2
  17. 甘氨酸
  18. Triton
  19. HCl
  20. 4x加载缓冲区
  21. 生物素淬灭溶液(参见配方)
  22. PBS/CaCl 2 2/MgCl 2 2(参见配方)
  23. IP缓冲区(参见配方)
  24. 库存(见配方)

设备

  1. 0.22μm过滤器
  2. 12孔板
  3. Western印迹设备

程序

  1. 制备PLO溶液:将90mg PLO加入180ml硼酸盐缓冲液(将1.668g硼酸溶解在180ml H 2 O中,并用NaOH调节pH至8.3),并通过0.22μm过滤器过滤灭菌。
  2. 将12孔板用PLO溶液在室温下在培养罩中过夜(1ml /孔)。 在细胞接种前第二天用无菌水清洗三次。
  3. 将HEK293细胞置于聚鸟氨酸包被的12孔板上,并根据需要进行转染
  4. 在细胞收获的当天,在0.5mg/ml的PBS/CaCl 2/MgCl 2溶液中溶解Sulfo-NHS-SS-生物素。 并存储在冰上
  5. 用1ml冰冷的PBS/CaCl 2/MgCl 2洗涤细胞两次。
  6. 生物素化
    1. 加入0.3ml 0.5mg/ml Sulfo-NHS-SS-生物素溶液
    2. 在冰上孵育30分钟,偶尔摇动
  7. 用冰冷的淬灭溶液洗涤三次,并在每次除去溶液前孵育5分钟,以中和未交联的试剂。
  8. 用120μlIP缓冲液溶解细胞,并在冰上孵育10分钟
  9. 将每个孔的所有内容物转移到1.5ml管中,并在4℃台式离心机上以Vmax(约14,000rpm)旋转10分钟。
  10. 保存10μl上清液(加入10μl4x加样缓冲液)作为供应 将100μl上清液与50μl固定化中性链亲和素的50%浆液混合,并在4℃下旋转2-3小时。
  11. 在4℃离心机中以5,000rpm旋转1分钟。 除去上清液,用1ml IP缓冲液通过在4℃室中旋转混合物5分钟洗涤珠子。 共洗涤三次。
  12. 最后一次洗涤后,取出上清液,加入25μl4x上样缓冲液的珠子。 充分混合并将管置于65℃水浴中10分钟以使蛋白质变性(选择65℃,因为我们发现在沸水中,许多膜蛋白形成聚集体,这导致蛋白质迁移到丙烯酰胺凝胶中的失败 )。 加载10μl的蛋白质印迹分析

食谱

  1. 生物素淬灭溶液(pH7.4) 50mM甘氨酸(0.1877g)的50ml PBS/CaCl 2/MgCl 2溶液/
  2. PBS/CaCl 2/MgCl 2
    PBS(+ 2.5mM CaCl 2,1mM MgCl 2,pH7.4)中的至少一种。
  3. IP缓冲液(pH 7.4)
    PBS

    50 ml
    EDTA
    5 mM
    0.5ml×0.5M(pH7.4)
    EGTA
    5 mM
    0.5ml×0.5M(pH7.4)
    NaPyrophophate
    10 mM
    0.223克
    NaF
    50 mM
    0.1 g
    NaVO 3    
    1 mM   
    0.5ml×100mM
    1%Triton
    1片蛋白酶抑制剂
  4. 股票
    1. 0.5M EDTA(pH7.4) 93.05g EDTA·2H 2 O·h/v 500ml ddH 2 O
      加入NaOH直至EDTA溶解,加入HCl调节pH至7.4
    2. 0.5 M EGTA(pH 7.4)
      95.1g EGTA·2H 2 O·
      500ml ddH 2 O
      加入NaOH直至EDTA溶解,加入HCl调节pH至7.4
    3. 100mM Na 3+ VO 4
      1.84g Na 3+ VO 4+ 10 H 2 O O 2/加入HCl至pH小于10(黄色溶液)。 将溶液煮沸至澄清无色。
    4. 10ml等分试样并在-20℃下冷冻

致谢

该协议以前在Huang等人(2006)中使用并改编自Huang等人。

参考文献

  1. Huang,G.N.,Zeng,W.,Kim,J.Y.,Yuan,J.P.,Han,L.,Muallem,S.and Worley,P.F。(2006)。 STIM1羧基末端激活天然SOC,I(crac)和TRPC1通道。 < et al。Nat Cell Biol 8(9):1003-1010。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Huang, G. N. (2012). Biotinylation of Cell Surface Proteins. Bio-protocol 2(9): e170. DOI: 10.21769/BioProtoc.170.
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