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β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation)
β1 整合素细胞表面免疫共沉淀(选择性免疫共沉淀)   

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Abstract

Immunoprecipitation (IP) is a widely used method to isolate a specific protein from a mixed protein sample using an antibody that exclusively binds to that particular protein. This technique allows studying protein-protein and protein-nucleic acid interactions or to identify post-translational protein modifications. Many proteins, in particular cell surface receptors, localize to different compartments within cells where they elicit distinct functions by interacting with specific proteins. Integrins represent a major family of cell surface receptors consisting of non-covalently associated α and β subunits that mediate the interaction of cells with their environment. However, integrins do not only localize to the cell surface but are also present in other compartments including the endoplasmic reticulum and endosomes where they engage with a distinct set of interacting partners or show distinct post-translational modifications. Standard immunoprecipitation of β1 integrins from a cell lysate without prior fractionation isolates β1 integrins from all compartments. In contrast, selective immunoprecipitation of cell surface β1 integrin allows enriching for the pool of β1 integrin on the cell surface thereby minimizing contaminations with β1 integrins from other subcellular compartments. To achieve this, living cells are incubated with a β1 integrin-specific antibody on ice to label cell surface β1 integrins prior to cell lysis and precipitation.

Keywords: Integrin(整合素), Immunoprecipitation(免疫共沉淀), Selective Immunoprecipitation(选择性沉淀)

Materials and Reagents

  1. Mouse fibroblasts lacking β1 integrin (β1 -/-) or re-expressing wild-type β1 integrin (β1 wt)
    Note: These cells are home-made immortalized mouse fibroblasts derived from floxed β1 parental cells. β1 -/- cells are used as negative control. However, the protocol can also be transferred to other mouse cell lines. When cells lacking β1 integrin are not available as negative control one has to include an unrelated antibody (see steps 2 and 4) to monitor for unspecific binding.
  2. Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX-I (Gibco, catalog number: 31966-021 )
  3. Fetal Bovine Serum (FBS) (PAA, catalog number: A15-101 )
  4. PBS (Sigma-Aldrich, catalog number: P4417 )
  5. 0.5% Trypsin/EDTA (Life Technologies, Gibco®, catalog number: 15400-054 )
  6. Primaquine bisphosphate (Sigma-Aldrich, catalog number: 160393 )
  7. Protein G sepharose (Protein G sepharose Fast Flow) (Sigma-Aldrich, catalog number: P3296 )
  8. BCA Protein Assay (Thermo Fisher Scientific, catalog number: 23227 )
  9. Triton X100
  10. Tris-HCl
  11. Na-deoxycholate
  12. SDS
  13. Glycerol
  14. Bromphenol blue
  15. Mercapthoethanol
  16. Protease inhibitors (Complete Mini EDTA-free) (Roche, catalog number: 04 693 159 001 )
  17. Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3) (Sigma-Aldrich, catalog numbers: P5726 and P0044 )
  18. Talin-1 antibody (1:1,000 for western blotting) (Sigma-Aldrich, catalog number: T3287 )
  19. SNX17 antibody (1:1,000 for western blotting) (Proteintech, catalog number: 10275-1-AP )
  20. β1 integrin IP buffer (see Recipes)
  21. 2x Laemmli sample buffer (see Recipes)

Equipment

  1. Cell scraper
  2. 10 cm cell culture dish
  3. Centrifuge
  4. 37 °C, 5% CO2 cell culture incubator
  5. 26-G needle attached to 1-ml syringe
  6. Heating block (Eppendorf Thermomixer compact or equivalent)

Procedure

  1. Wash mouse fibroblasts expressing β1 integrin (β1 wt) and fibroblasts lacking β1 integrin (β1 -/-) with PBS, trypsinize and count cells using Glass slide with grids.
  2. For both cell lines plate 2 x 106 cells per 10 cm dish and incubate in DMEM/10%FBS in the 5% CO2 incubator at 37 °C overnight.
    Note: We used the cell line lacking β1 integrin as negative control. Alternatively, one can plate cells expressing β1 integrin for incubation with an unrelated control antibody (step 4).
  3. Place dishes on ice and wash twice with ice-cold PBS (4 ml/dish).
  4. To label cell surface β1 integrin, incubate the cells in 3 ml ice-cold DMEM/10%FBS containing the anti-β1 integrin antibody or an unrelated antibody as negative control.
    Note: Anti-β1 integrin antibody: The antibody has to be directed against an epitope in the extracellular domain of integrin and has to recognize β1 integrin in its native conformation. We used a home-made antibody against mouse β1 integrin in a concentration of 1:1,500 (Bottcher et al., 2012). For other antibodies the amount has to be determined experimentally.
    Control antibody: Should be derived from the same species as the anti-β1 integrin antibody and should be used in the same concentration.
  5. Place dishes on rocker (approximately 7 see-saw movements per minute) at 4 °C and incubate for 60 minutes.
  6. Place dishes on ice and wash twice with ice-cold PBS (4 ml/dish).
    To selectively immunoprecipitate cell surface β1 integrin continue with step 9.
  7. To immunoprecipitate β1 integrin from the endosomal compartment, incubate the cells in DMEM/10%FBS containing primaquine for 15 min at 37 °C.
    Note: After antibody binding to cell surface β1 integrins on ice it is possible to induce β1 integrin endocytosis by incubating the cells at 37 °C. This enables the antibody-β1 integrin complexes to reach the endosomal compartment. β1 integrins are rapidly internalized and the addition of primaquine inhibits recycling of β1 integrin from endosomes back to the cell surface thereby enriching the amount of β1 integrin in endosomes. Depending on the cell type, 0.6 μM to 0.6 mM primaquine are used.
  8. Place dishes on ice and wash once with ice-cold PBS (4 ml/dish).
  9. Lyse cells in 1 ml β1 integrin IP buffer per 10 cm dish for 15 min on ice.
  10. Scrape off cells and transfer the cell lysate into pre-cooled 1.5 ml reaction tubes.
  11. Sonicate briefly or pass several times through a 26 gauge needle.
  12. Spin cell lysate at 17,000 x g for 10 min at 4 °C.
  13. Transfer the supernatant into fresh pre-cooled 1.5 ml reaction tubes.
  14. Take out 60 μl for whole cell lysate sample and 5.0 μl to determine the protein concentration.
  15. Take 30.0 μl Protein G sepharose slurry per 1 mg protein, wash three times with β1 integrin IP buffer and add equal amount of the cell lysis supernatant per sample (between 1.0-1.5 mg cell lysate was used per sample).
  16. Incubate for 2 hours at 4 °C on a rocking platform or a rotator.
  17. Spin the Eppendorf tube at 1,500 x g for 2 min at 4 °C. Remove the supernatant completely and wash the beads 3-5 times with 500 μl of β1 integrin IP buffer.
  18. After the last wash, take off supernatant and elute proteins by heating to 90-100 °C for 7 minutes in 60 μl of 2x Laemmli sample buffer.
  19. Spin at 10,000 x g for 30 sec, collect supernatant and load onto the gel. Supernatant samples can be collected and kept frozen at this point if the gel is to be run later.
    Note: β1 integrin translation/processing is a tightly controlled step-wise process that starts with the synthesis of a 88 kDa polypeptide that undergoes sequential glycosylation in the ER (‘immature’ form 105 kDa) and in the Golgi giving rise to incompletely glycosylated β1 integrin subunit and a complete or ‘mature’ β1 subunit of around 125 kDa.
    A successful immunoprecipitation of the cell-surface β1 integrins can be shown by western blotting with an antibody against β1 integrin (e.g. the antibody used for immunoprecipiation; dilution 1:10,000). The immature 105 kDa β1 integrin should be strongly reduced, ideally not detectable, after the immunoprecipitation. To further characterize the purity of your cell surface β1 integrins, the precipitate can be analyzed by western blotting for co-immunoprecipitated proteins such as talin-1 (interacts with β1 integrin at the plasma membrane; 1:1,000 for western blotting) or SNX17 (interacts with β1 integrin on endosomes; 1:1,000 for western blotting).

Recipes

  1. β1 integrin IP buffer
    50 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    1% Triton X100
    0.1% Na-deoxycholate
    1 mM EDTA
    Protease inhibitors (Complete Mini EDTA-free; 1 tablet for 10 ml of buffer)
    Phosphatase inhibitors (Phosphatase Inhibitor Cocktail 2 and 3; 1:100 dilution from stock)
  2. 2x Laemmli sample buffer
    120 mM Tris-HCl (pH 6.8)
    4% SDS
    20% glycerol
    4 mM EDTA
    0.001% bromphenol blue
    2% mercapthoethanol

Acknowledgments

This protocol was adapted from a paper by Böttcher et al. (2012). We thank R. Fässler for critically reading the manuscript and continuous support. This work was funded by the Deutsche Forschungsgemeinschaft (SFB 914, project A05).

References

  1. Bottcher, R. T., Stremmel, C., Meves, A., Meyer, H., Widmaier, M., Tseng, H. Y. and Fassler, R. (2012). Sorting nexin 17 prevents lysosomal degradation of β1 integrins by binding to the β1-integrin tail. Nat Cell Biol 14(6): 584-592.

简介

免疫沉淀(IP)是一种广泛使用的方法,使用仅与该特定蛋白结合的抗体从混合蛋白样品中分离特定蛋白。这种技术允许研究蛋白质 - 蛋白质和蛋白质 - 核酸相互作用或识别翻译后蛋白质修饰。许多蛋白质,特别是细胞表面受体,定位于细胞内的不同区室,其中它们通过与特定蛋白质相互作用引起不同的功能。整联蛋白代表由非共价缔合的α和β亚基组成的细胞表面受体的主要家族,其介导细胞与其环境的相互作用。然而,整联蛋白不仅定位于细胞表面,而且存在于其它区室中,包括内质网和内体,其中它们与不同组的相互作用配偶体接合或显示不同的翻译后修饰。来自细胞裂解物的β1整联蛋白的标准免疫沉淀,没有预先分级分离来自所有区室的β1整联蛋白。相比之下,细胞表面β1整合素的选择性免疫沉淀允许富集β1整联蛋白在细胞表面上的库,从而使来自其他亚细胞区室的β1整联蛋白的污染最小化。为了实现这一点,活细胞与β1整合素特异性抗体在冰上孵育以在细胞裂解和沉淀之前标记细胞表面β1整联蛋白。

关键字:整合素, 免疫共沉淀, 选择性沉淀

材料和试剂

  1. 缺乏β1整联蛋白(β1 -/- )或重新表达野生型β1整联蛋白(β1重)的小鼠成纤维细胞
    注意:这些细胞是来自floxedβ1亲本细胞的自制永生化小鼠成纤维细胞。 β1 -/- 细胞用作阴性对照。 然而,该方案也可以转移到其他小鼠细胞系。 当缺乏β1整联蛋白的细胞不可用作阴性对照时,必须包括不相关的抗体(参见步骤2和4)以监测非特异性结合。
  2. 含有GlutaMAX-I(Gibco,目录号:31966-021)的Dulbecco改良的Eagle培养基(DMEM)
  3. 胎牛血清(FBS)(PAA,目录号:A15-101)
  4. PBS(Sigma-Aldrich,目录号:P4417)
  5. 0.5%胰蛋白酶/EDTA(Life Technologies,Gibco ,目录号:15400-054)
  6. 二磷酸伯氨喹(Sigma-Aldrich,目录号:160393)
  7. 蛋白G琼脂糖(Protein G sepharose Fast Flow)(Sigma-Aldrich,目录号:P3296)
  8. BCA蛋白测定(Thermo Fisher Scientific,目录号:23227)
  9. Triton X100
  10. Tris-HCl
  11. Na-脱氧胆酸盐
  12. SDS
  13. 甘油
  14. 溴酚蓝
  15. 巯基乙醇
  16. 蛋白酶抑制剂(Complete Mini EDTA-free)(Roche,目录号:04 693 159 001)
  17. 磷酸酶抑制剂(磷酸酶抑制剂混合物2和3)(Sigma-Aldrich,目录号:P5726和P0044)
  18. Talin-1抗体(1:1,000,用于western印迹)(Sigma-Aldrich,目录号:T3287)
  19. SNX17抗体(用于western印迹的1:1,000)(Proteintech,目录号:10275-1-AP)
  20. β1整合素IP缓冲液(参见配方)
  21. 2x Laemmli样品缓冲液(参见配方)

设备

  1. 细胞刮刀
  2. 10厘米细胞培养皿
  3. 离心机
  4. 37℃,5%CO 2细胞培养箱中培养
  5. 26-G针连接到1 ml注射器
  6. 加热块(Eppendorf Thermomixer compact或同等品)

程序

  1. 用PBS洗涤表达β1整合素(β1wt)的小鼠成纤维细胞和缺乏β1整合素(β1 -/- )的成纤维细胞,胰蛋白酶消化并使用具有格栅的玻璃载玻片计数细胞。
  2. 对于两个细胞系板,每10cm皿2×10 6个细胞,并在37℃下在5%CO 2培养箱中的DMEM/10%FBS中孵育过夜。 br /> 注意:我们使用缺乏β1整合素的细胞系作为阴性对照。或者,可以将表达β1整联蛋白的细胞与无关的对照抗体一起温育(步骤4)。
  3. 将菜放在冰上,用冰冷的PBS(4ml /皿)洗两次
  4. 为了标记细胞表面β1整合素,将细胞在含有抗β1整联蛋白抗体或不相关抗体的3ml冰冷的DMEM/10%FBS中孵育作为阴性对照。
    注意:抗β1整联蛋白抗体:抗体必须针对整联蛋白的胞外结构域中的表位,并且必须以其天然构象识别β1整联蛋白。我们使用针对小鼠β1整合素的自制抗体,浓度为1:1,500(Bottcher等,2012)。对于其他抗体,该量必须通过实验确定。
    对照抗体:应来自与抗β1整联蛋白抗体相同的物种,并应以相同的浓度使用。
  5. 在4℃下将菜肴放在摇床上(每分钟约7次跷跷板运动),并孵育60分钟
  6. 将菜放在冰上,用冰冷的PBS(4ml /皿)洗两次 选择性免疫沉淀细胞表面β1整合素继续步骤9.
  7. 为了从内体室免疫沉淀β1整合素,在37℃下将细胞在含有伯氨喹的DMEM/10%FBS中孵育15分钟。
    注意:在冰上抗体与细胞表面β1整合素结合后,通过在37℃下孵育细胞可以诱导β1整合素内吞。这使得抗体-β1整联蛋白复合物能够到达内体区室。 β1整联蛋白被快速内化,并且伯氨喹的添加抑制β1整联蛋白从内体回到细胞表面的再循环,从而富集内体中β1整联蛋白的量。根据细胞类型,使用0.6μM至0.6mM的primaquine。
  8. 将盘放在冰上,用冰冷的PBS(4ml /皿)洗一次
  9. 溶解细胞在1mlβ1整合素IP缓冲液每10厘米培养皿在冰上15分钟
  10. 刮去细胞并将细胞裂解物转移到预冷却的1.5ml反应管中
  11. 用26号针短暂超声处理或通过几次。
  12. 在4℃下,以17,000×g离心10分钟,旋转细胞裂解物
  13. 将上清液转移到新鲜的预冷却的1.5ml反应管中
  14. 取出60微升全细胞裂解物样品和5.0微升以确定蛋白质浓度
  15. 取每30 mg蛋白G Sepharose浆液30.0μl,用β1整合素IP缓冲液洗涤三次,每个样品加入等量的细胞裂解上清液(每个样品使用1.0-1.5 mg细胞裂解液)。
  16. 在4℃下在摇摆平台或旋转器上孵育2小时
  17. 在4℃下以1500xg转动Eppendorf管2分钟。完全除去上清液,用500μl的β1整合素IP缓冲液洗珠子3-5次
  18. 最后一次洗涤后,取出上清液并通过在60μl2×Laemmli样品缓冲液中加热至90-100℃洗脱蛋白质7分钟。
  19. 在10,000×g下旋转30秒,收集上清液并加载到凝胶上。如果凝胶稍后运行,可以收集上清液样品并保持冷冻。
    注意:β1整联蛋白翻译/加工是严格控制的逐步过程,其开始于88kDa多肽的合成,其在ER中("未成熟"形式105kDa)和在高尔基体中经历连续糖基化不完全糖基化的β1整合素亚基和约125kDa的完全或"成熟"β1亚基。
    可以通过用针对β1整联蛋白的抗体(例如用于免疫沉淀的抗体;稀释1:10,000)的western印迹显示细胞表面β1整联蛋白的成功的免疫沉淀。在免疫沉淀后,未成熟的105kDaβ1整联蛋白应该被强烈还原,理想地不可检测。为了进一步表征细胞表面β1整合素的纯度,可以通过蛋白质印迹分析沉淀物用于共免疫沉淀的蛋白质,例如talin-1(在质膜处与β1整联蛋白相互作用;对于蛋白质印迹为1:1,000)或SNX17与内体上的β1整合素相互作用; 1:1,000用于western印迹)。

食谱

  1. β1整合素IP缓冲液
    50mM Tris-HCl(pH7.5) 150mM NaCl 1%Triton X100
    0.1%脱氧胆酸钠 1mM EDTA
    蛋白酶抑制剂(Complete Mini EDTA-free; 1片用于10ml缓冲液) 磷酸酶抑制剂(磷酸酶抑制剂混合物2和3;从库存稀释1:100)
  2. 2x Laemmli样品缓冲液
    120 mM Tris-HCl(pH 6.8)
    4%SDS
    20%甘油 4mM EDTA
    0.001%溴酚蓝
    2%巯基乙醇

致谢

该协议改编自Böttcher等人的论文(2012)。 我们感谢R.Fässler批判性地阅读手稿和持续的支持。 这项工作由德意志交易所(SFB 914,项目A05)资助。

参考文献

  1. Bottcher,R.T.,Stremmel,C.,Meves,A.,Meyer,H.,Widmaier,M.,Tseng,H.Y.and Fassler,R。(2012)。 对nexin 17进行排序可以通过结合β1整合素尾部来防止β1整合素的溶酶体降解。 Nat Cell Biol 14(6):584-592。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Böttcher, R. T. (2013). β1 Integrin Cell-surface Immunoprecipitation (Selective Immunoprecipitation). Bio-protocol 3(21): e962. DOI: 10.21769/BioProtoc.962.
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