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In vitro Lipid Binding Assay
体外脂质结合试验   

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Abstract

This is a protocol to examine in vitro protein-lipid binding using membrane strips coated with various lipids. It has been successfully used to study in vitro interaction between lipids and C. elegans proteins.

Materials and Reagents

  1. Membrane Lipid strip (Echelon Bioscience, catalog number: P-6001 )
  2. Proteins of interest (EGFP-NRF-5-Flag, EGFP-Flag as control)
  3. Anti-Flag M2 antibody (Sigma-Aldrich, catalog number: A2220 )
  4. Primary antibodies (Anti-Flag M2 from Mouse, sigma, catalog number: F1804 )
  5. HRP-conjugated secondary antibodies (e.g. Goat anti-Mouse LgG (H+L) HRP, The Jackson Laboratory, catalog number: 115-035-003 )
  6. SuperSignal West Pico reagent (Thermo Fisher Scientific, catalog number: 34080 )
  7. 293T cells
  8. Tris-Cl
  9. NaCl
  10. Tween-20
  11. Ca2+ chloride
  12. Zn2+ sulfate
  13. Non-fat milk
  14. Blocking buffer (see Recipes)
  15. Incubation buffer (see Recipes)
  16. Wash buffer (see Recipes)

Equipment

  1. Vortexer
  2. Rotator

Procedure

  1. Membrane strips coated with various lipids are incubated in 10 ml of blocking buffer for 1 h at room temperature in dark (the commercially available membrane strip has been coated with lipids).
  2. 20-60 μg of proteins are added to 6 ml incubation buffer with membrane strips and incubated overnight at 4 °C. In our study, we used Flag-tagged protein purified from 293T cells (Zhang et al., 2012). However, proteins purified from other sources can all be used. Negative controls should be included according to specific proteins used in this assay.
  3. Membrane strips are washed extensively with wash buffer for 3 times at RT, 10 min each wash on rotator.
  4. Membrane strips with bounded proteins are incubated with primary antibodies (Anti-Flag M2 from mouse, 1:1,000) in incubation buffer for 3 h at RT.
  5. Wash 3 times at RT with wash buffer, 10 min each wash on rotator.
  6. Incubate membrane strips with HRP-conjugated secondary antibodies (Goat anti-Mouse LgG (H+L) HRP, 1:10,000) in incubation buffer for 1 h at RT.
  7. Membranes are washed as above and protein-antibody interaction is detected using SuperSignal West Pico reagent.

Recipes

  1. Blocking buffer
    25 mM Tris-Cl
    150 mM NaCl
    0.1% Tween-20
    1% non-fat milk
  2. Incubation buffer
    25 mM Tris-Cl
    150 mM NaCl
    0.1% Tween-20
    1% non-fat milk
    2 mM Ca2+ chloride
    1 mM Zn2+ sulfate
  3. Wash buffer
    25 mM Tris-Cl
    150 mM NaCl
    0.1% Tween-20
    2 mM Ca2+ chloride
    1 mM Zn2+ sulfate

Acknowledgments

This protocol is adapted from Zhang et al. (2012) and Wang et al. (2010).

References

  1. Wang, X., Li, W., Zhao, D., Liu, B., Shi, Y., Chen, B., Yang, H., Guo, P., Geng, X., Shang, Z., Peden, E., Kage-Nakadai, E., Mitani, S. and Xue, D. (2010). Caenorhabditis elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor. Nat Cell Biol 12(7): 655-664.
  2. Zhang, Y., Wang, H., Kage-Nakadai, E., Mitani, S. and Wang, X. (2012). C. elegans secreted lipid-binding protein NRF-5 mediates PS appearance on phagocytes for cell corpse engulfment. Curr Biol 22(14): 1276-1284.

简介

这是使用涂覆有各种脂质的膜条来检查体外蛋白质 - 脂质结合的方案。 它已经成功地用于研究脂质和C之间的体外相互作用。 elegans蛋白质。

材料和试剂

  1. 膜脂质条(Echelon Bioscience,目录号:P-6001)
  2. 感兴趣的蛋白质(EGFP-NRF-5-Flag,EGFP- Flag作为对照)
  3. 抗Flag M2抗体(Sigma-Aldrich,目录号:A2220)
  4. 一抗(来自Mouse,Sigma的目录号:F1804的抗Flag M2)
  5. HRP缀合的二抗(例如,山羊抗小鼠LgG(H + L)HRP,The Jackson Laboratory,目录号:115-035-003)
  6. SuperSignal West Pico试剂(Thermo Fisher Scientific,目录号:34080)
  7. 293T细胞
  8. Tris-Cl
  9. NaCl
  10. 吐温-20
  11. Ca <2> 氯化物
  12. Zn <2>硫酸盐
  13. 脱脂牛奶
  14. 阻止缓冲区(参见配方)
  15. 孵育缓冲液(参见配方)
  16. 洗涤缓冲液(见配方)

设备

  1. Vortexer
  2. 旋转器

程序

  1. 包被有各种脂质的膜条在室温下在黑暗中(市售的膜条已经用脂质包被)在10ml封闭缓冲液中孵育1小时。
  2. 将20-60μg蛋白质加入到具有膜条的6ml孵育缓冲液中,并在4℃下孵育过夜。 在我们的研究中,我们使用从293T细胞纯化的Flag标记的蛋白质(Zhang等人,2012)。 然而,从其他来源纯化的蛋白质都可以使用。 根据本测定中使用的特定蛋白质,应包括阴性对照
  3. 将膜条用洗涤缓冲液在室温下彻底洗涤3次,每次在旋转器上洗涤10分钟
  4. 具有结合蛋白的膜条带在第一抗体(来自小鼠的抗Flag M2,1:1,000)在孵育缓冲液中在室温孵育3小时。
  5. 在室温下用洗涤缓冲液洗涤3次,每次在旋转器上洗涤10分钟
  6. 用孵育缓冲液中的HRP缀合的第二抗体(山羊抗小鼠LgG(H + L)HRP,1:10,000)在室温孵育膜条1小时。
  7. 如上所述洗涤膜,并使用SuperSignal West Pico试剂检测蛋白质 - 抗体相互作用

食谱

  1. 阻塞缓冲区
    25mM Tris-Cl 150mM NaCl 0.1%Tween-20
    1%脱脂牛奶
  2. 孵育缓冲区
    25mM Tris-Cl 150mM NaCl 0.1%Tween-20
    1%脱脂牛奶
    2mM Ca 2+ + 氯化物 1mM Zn 2+硫酸盐
  3. 洗涤缓冲液
    25mM Tris-Cl 150mM NaCl 0.1%Tween-20
    2mM Ca 2+ + 氯化物 1mM Zn 2+硫酸盐

致谢

该协议改编自Zhang等人(2012)和Wang等人(2010)。

参考文献

  1. Wang,X.,Li,W.,Zhao,D.,Liu,B.,Shi,Y.,Chen,B.,Yang,H.,Guo,P.,Geng,X.,Shang,Z。 Peden,E.,Kage-Nakadai,E.,Mitani,S。和Xue,D。(2010)。 秀丽隐杆线虫转甲状腺素样蛋白TTR-52介导凋亡细胞的识别 通过CED-1吞噬细胞受体。 Nat Cell Biol 12(7):655-664。
  2. Zhang,Y.,Wang,H.,Kage-Nakadai,E.,Mitani,S.and Wang,X.(2012)。 C。 线虫分泌的脂质结合蛋白NRF-5介导吞噬细胞上的PS出现,用于细胞吞噬。 Curr Biol 22(14):1276-1284
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhang, Y. and Wang, X. (2013). In vitro Lipid Binding Assay. Bio-protocol 3(9): e694. DOI: 10.21769/BioProtoc.694.
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