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Cell Surface Protein-protein Binding on COS-7 Cells
COS-7 细胞表面蛋白质之间的结合   

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参见作者原研究论文

本实验方案简略版
Neuron
May 2013

Abstract

Examination of interactions between a transmembrane protein and a soluble protein by pull-down or immunoprecipitation assays can be tricky and complicated due to the detergent extraction of membrane proteins during the lysate preparation step. The choice and concentration of detergents must be determined empirically and the procedure can be burdensome. Here, we describe a simplified binding assay by expressing the membrane protein of interest in COS-7 cells and applying detergent-free solutions containing an extracellular protein to be tested. The binding is then examined by immunocytochemistry.

Keywords: Membrane protein (膜蛋白), Protein-protein interaction (蛋白质-蛋白质相互作用), Surface binding (表面结合)

Materials and Reagents

  1. COS-7 cells (ATCC, catalog number: CRL-1651 TM)
  2. A mammalian expression plasmid encoding a membrane protein of interest
  3. A plasmid encoding an extracellular protein fused to the Fc portion of the human IgG [for example, pFUSE series by InvivoGen and pSXFc in Eshed et al. (2005) are options for cloning vectors to make this fusion construct]
  4. Dulbecco's modified Eagle Medium (DMEM), high glucose, no glutamine (Life Technologies, catalog number: 11960-044 )
  5. FetalClone III serum (Thermo Fisher Scientific, catalog number: SH30109.03 )
  6. 100x GlutaMAX-I (Life Technologies, catalog number: 35050-061 )
  7. Lipofectamine 2000 (Life Technologies, catalog number: 11668-019 )
  8. Dulbecco's phosphate-buffered saline (DPBS) (no calcium, no magnesium) (Life Technologies, catalog number: 14190-144 )
  9. Virus production serum-free medium (VP-SFM) (Life Technologies, catalog number: 11681-020 )
  10. 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H4034 )
  11. Protein Concentrators (9K MWCO, 7 ml) (Thermo Fisher Scientific, catalog number: 87748 )
  12. Goat anti-human IgG, Fcγ fragment specific, conjugated with a fluorophore (e.g. Jackson ImmunoResearch Laboratories, catalog number: 109-545-098 for Fluorescein and 109-585-098 for Texas Red)
  13. COS-7 culture medium (see Recipes)
  14. 0.2 M PB (see Recipes)
  15. 4% PFA (see Recipes)
  16. PBS (see Recipes)

Equipment

  1. 6-well cell culture plates (Corning Incorporated, catalog number: 3516 )
  2. Glass coverslips (VWR International, catalog number: 48366-227 )
  3. 10-cm dish
  4. Centrifuge
  5. 37 °C/5% CO2 cell culture incubator
  6. A regular fluorescence microscope

Procedure

  1. Production of the medium containing the extracellular protein.
    1. Seed COS-7 cells in a 10-cm dish.
    2. Transfect the cells with the plasmid encoding the extracellular protein fused to the human IgG Fc (we use Lipofectamine 2000: 1 μg of DNA + 2 μl of Lipofectamine 2000 for one well in a 6-well plate, and follow the manufacturer’s instruction).
    3. One day after transfection, remove all the medium, wash the culture with 6 ml of DPBS twice and add 10 ml of VP-SFM containing 2x GlutaMAX-I.
    4. Two days later, collect all the medium and add 10 ml of fresh VP-SFM/2x GlutaMAX-I to the culture.
    5. Centrifuge the harvested medium at 3,000 x g for 3 min, collect the supernatant and neutralize it by adding to a final concentration 10 mM HEPES-Na+, pH 8.0.
    6. Repeat steps 1d and 1e.
    7. Pool two batches of the medium and concentrate with a protein concentrator to 1/10x the original volume. The concentrated medium can be stored at -80 °C(we usually store the concentrated medium in 1-ml aliquots. For our fusion proteins, the binding activity is still good even after 5 freeze-thaw cycles. If freezing and thawing are a concern, it is recommended to first test the binding activity from unfrozen preparations).
  2. Seed COS-7 cells directly into the 6-well plates containing glass coverslips (no treatment required) and the culture medium.
  3. Transfect the cells with the plasmid encoding the membrane protein.
  4. Two days later, thaw the concentrated medium containing the Fc fusion protein from step 1.
  5. For the binding assay on one coverslip of cells, take 100 μl of the concentrated medium and add the goat anti-human Fc antibodies conjugated with a fluorophore at a final concentration of 7.5 ng/ml. Incubate on ice for 30 min (cover with foil to protect the fluorophore from the light).
  6. Transfer the coverslips with cells from the original 6-well plate to a new empty 6-well plate and add the mixture from step 5 onto the coverslips carefully (the 100-μl mixture stays on the top of the coverslip.) Incubate at room temperature for 30 min (cover with foil).
  7. Remove the mixture of the concentrated medium and goat anti-human Fc antibodies and rinse with 2 ml of ice-cold DPBS three times.
  8. Fix the cells on coverslips and follow the regular immunostaining procedure. The fixation and staining conditions depend on the properties of the primary and secondary antibodies. For most of our antibodies, the following procedure is performed: (protect from light for the whole procedure).
    1. Add 2 ml of ice-cold 4% PFA per well and incubate for 20 min at 4 °C.
    2. Rinse with 2 ml of room-temperature PBS three times.
    3. The cells on the coverslips are ready for blocking/permeabilizing and immunostaining to visualize the expression of the membrane protein.


    Figure 1. An example of the binding assay. COS-7 cells were transfected with an NF186-expressing plasmid and then incubated with a mixture of goat anti-human Fc antibodies and  concentrated medium containing Fc or BcG3-Fc. The nuclei were visualized by Hoechst. Fc does not interact with NF186 and is the negative control. BcG3-Fc specifically interacts with NF186 and does not bind to untransfected cells. Scale bar = 16 μm.

Recipes

  1. COS-7 culture medium (100 ml)
    89 ml of DMEM
    10 ml of FetalClone III
    1 ml of 100x GlutaMAX-I
    Stored at 4 °C
  2. 0.2 M PB (1L)
    Dissolve 4.56 g of NaH2PO4 and 23.004 g of Na2HPO4 in 900 ml of ddH2O and adjust to 1,000 ml
    Stored at room temperature
  3. 4% PFA (100 ml)
    Dissolve 4 g of paraformaldehyde in 50 ml of 50-60 °C ddH2O by adding a few drops of 10 N NaOH
    Add 50 ml of 0.2 M PB
    Adjust pH to 7.2-7.4 by adding concentrated HCl
    Good for 2 days if stored at 4 °C
  4. PBS
    150 mM NaCl
    10.4 mM sodium phosphate (pH 7.2)

Acknowledgments

This protocol was adapted from Eshed et al. (2005) and Susuki et al. (2013).

References

  1. Eshed, Y., Feinberg, K., Poliak, S., Sabanay, H., Sarig-Nadir, O., Spiegel, I., Bermingham Jr, J. R. and Peles, E. (2005). Gliomedin mediates Schwann cell-axon interaction and the molecular assembly of the nodes of Ranvier. Neuron 47(2): 215-229. 
  2. Susuki, K., Chang, K.-J., Zollinger, D. R., Liu, Y., Ogawa, Y., Eshed-Eisenbach, Y., Dours-Zimmermann, M. T., Oses-Prieto, J. A., Burlingame, A. L. and Seidenbecher, C. I. (2013). Three mechanisms assemble central nervous system nodes of ranvier. Neuron 78(3): 469-482. 

简介

通过下拉或免疫沉淀测定法检查跨膜蛋白和可溶性蛋白质之间的相互作用可能是棘手和复杂的,因为在裂解物制备步骤期间膜蛋白的去污剂提取。 洗涤剂的选择和浓度必须根据经验确定,并且该方法可能是繁重的。 在这里,我们描述简化的绑定测定通过表达感兴趣的COS蛋白的COS 7细胞和应用含有待测试的细胞外蛋白的无洗涤剂溶液。 然后通过免疫细胞化学检查结合。

关键字:膜蛋白, 蛋白质-蛋白质相互作用, 表面结合

材料和试剂

  1. COS-7细胞(ATCC,目录号:CRL-1651 )
  2. 编码感兴趣的膜蛋白的哺乳动物表达质粒
  3. 编码与Fc部分融合的胞外蛋白的质粒 人IgG [例如,InvivoGen的pFUSE系列和Eshed等人(2005)的pSXFc是用于克隆载体以制备该融合构建体的选择]
  4. Dulbecco改良的Eagle培养基(DMEM),高葡萄糖,无谷氨酰胺(Life Technologies,目录号:11960-044)
  5. 胎牛血清III血清(Thermo Fisher Scientific,目录号:SH30109.03)
  6. 100x GlutaMAX-I(Life Technologies,目录号:35050-061)
  7. Lipofectamine 2000(Life Technologies,目录号:11668-019)
  8. Dulbecco磷酸盐缓冲盐水(DPBS)(无钙,无镁)(Life Technologies,目录号:14190-144)
  9. 病毒产生无血清培养基(VP-SFM)(Life Technologies,目录号:11681-020)
  10. 4-(2-羟乙基)哌嗪-1-乙磺酸(HEPES)(Sigma-Aldrich,目录号:H4034)
  11. 蛋白质浓缩器(9K MWCO,7ml)(Thermo Fisher Scientific,目录号:87748)
  12. 山羊抗人IgG,Fcγ片段特异性,与荧光团缀合(例如Jackson ImmunoResearch Laboratories,目录号:109-545-098用于荧光素,109-585-098用于德克萨斯红) >
  13. COS-7培养基(见配方)
  14. 0.2 M PB(参见配方)
  15. 4%PFA(参见配方)
  16. PBS(请参阅配方)

设备

  1. 6孔细胞培养板(Corning Incorporated,目录号:3516)
  2. 玻璃盖玻片(VWR International,目录号:48366-227)
  3. 10厘米平皿
  4. 离心机
  5. 37℃/5%CO 2细胞培养孵化器
  6. 普通荧光显微镜

程序

  1. 含有细胞外蛋白质的培养基的生产。
    1. 种子COS-7细胞在10厘米的培养皿
    2. 用编码与人IgG Fc融合的细胞外蛋白质的质粒(我们使用Lipofectamine 2000:1μgDNA +2μlLipofectamine 2000用于6孔板中的一个孔,并根据制造商的说明书)转染细胞。 br />
    3. 转染后一天,除去所有培养基,用6ml DPBS洗涤培养物两次,加入10ml含2×GlutaMAX-I的VP-SFM。
    4. 两天后,收集所有培养基,并向培养物中加入10ml新鲜的VP-SFM/2x GlutaMAX-I。
    5. 在3,000×g离心收获的培养基3分钟,收集上清液,通过加入终浓度10mM HEPES-Na + pH 8.0中和它。 >
    6. 重复步骤1d和1e。
    7. 池两批培养基,用蛋白浓缩器浓缩至原体积的1/10倍。浓缩培养基可以储存在-80℃(我们通常以1ml等分试样存储浓缩培养基。对于我们的融合蛋白,即使在5次冻融循环后,结合活性仍然良好,如果冷冻和解冻是关注的,建议首先测试未冻结制剂的结合活性)。
  2. 种COS-7细胞直接进入含有玻璃盖玻片(不需要处理)的6孔板和培养基。
  3. 用编码膜蛋白的质粒转染细胞
  4. 两天后,解冻来自步骤1的含有Fc融合蛋白的浓缩培养基
  5. 对于一个盖玻片上的结合测定,取100μl的浓缩培养基,并加入与荧光团缀合的山羊抗人Fc抗体,终浓度为7.5ng/ml。在冰上孵育30分钟(用箔覆盖,以保护荧光团免受光照)。
  6. 转移盖玻片从细胞从原始的6孔板到新的空6孔板,并将步骤5的混合物小心地加到盖玻片上(100μl混合物停留在盖玻片的顶部)。在室温下孵育30分钟(用铝箔覆盖)。
  7. 除去浓缩培养基和山羊抗人Fc抗体的混合物,并用2ml冰冷的DPBS冲洗三次。
  8. 将细胞固定在盖玻片上,并按照常规免疫染色程序。固定和染色条件取决于一抗和二抗的性质。对于我们的大多数抗体,进行以下程序:(对于整个程序避光)。
    1. 每孔加入2ml冰冷的4%PFA,并在4℃下孵育20分钟
    2. 用2ml室温PBS冲洗三次。
    3. 盖玻片上的细胞准备好阻断/透化和免疫染色以显现膜蛋白的表达。


    图1.结合测定的实例。用表达NF186的质粒转染COS-7细胞,然后与山羊抗人Fc抗体混合物一起温育, 含有Fc或BcG3-Fc的浓缩培养基。 通过Hoechst显现细胞核。 Fc不与NF186相互作用并且是阴性对照。 BcG3-Fc与NF186特异性相互作用,不与未转染的细胞结合。 比例尺= 16μm。

食谱

  1. COS-7培养基(100ml) 89 ml DMEM
    10ml胎牛血清III / 1ml的100×GlutaMAX-I / 储存在4°C
  2. 0.2 M PB(1L)
    在900ml ddH 2中溶解4.56g的NaH 2 PO 4和23.004g的Na 2 HPO 4, sub> 2 O,并调整到1000毫升
    在室温下贮存
  3. 4%PFA(100ml) 通过加入几滴10N NaOH将4g多聚甲醛溶解在50ml 50-60℃ddH 2 O中。
    加入50ml 0.2M PB
    加入浓HCl,调节pH至7.2-7.4 如果存储在4°C,则有效期为2天
  4. PBS
    150mM NaCl 10.4mM磷酸钠(pH7.2)

致谢

该方案改编自Eshed等(2005)和Susuki等人(2013)。

参考文献

  1. Eshed,Y.,Feinberg,K.,Poliak,S.,Sabanay,H.,Sarig-Nadir,O.,Spiegel,I.,Bermingham Jr,JR和Peles,E。(2005) http://www.ncbi.nlm.nih.gov/pubmed/16039564"target ="_ blank"> Gliomedin介导施万 细胞轴突相互作用和Ranvier的淋巴结的分子组装。 Neuron 47(2):215-229。 
  2. Susishi,K.,Chang,K.-J.,Zollinger,DR,Liu,Y.,Ogawa,Y.,Eshed-Eisenbach,Y.,Dours-Zimmermann,MT,Oses-Prieto,JA,Burlingame, Seidenbecher,CI(2013)。 三种机制组装了ranvier的中枢神经系统节点。 神经元 78(3):469-482。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chang, K. and Rasband, M. N. (2014). Cell Surface Protein-protein Binding on COS-7 Cells. Bio-protocol 4(1): e1020. DOI: 10.21769/BioProtoc.1020.
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