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Virus overlay assay is a method to detect protein-protein interaction in vitro. We performed the virus overlay assay to identify the receptor proteins interacting with the infectious spleen and kidney necrosis virus (ISKNV).

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Virus Overlay Assay (Far-Western blotting)
病毒覆盖检测(Far-Western蛋白杂交)

微生物学 > 微生物生物化学 > 蛋白质 > 免疫检测
作者: Kun-Tong Jia
Kun-Tong JiaAffiliation: School of Marine Sciences, Sun Yat-Sen University, Guangzhou, China
Bio-protocol author page: a874
Chang-Jun Guo
Chang-Jun GuoAffiliation: School of Marine Sciences, Sun Yat-Sen University, Guangzhou, China
For correspondence: lsshjg@mail.sysu.edu.cn
Bio-protocol author page: a875
Xiao-Bo Yang
Xiao-Bo YangAffiliation: School of Marine Sciences, Sun Yat-Sen University, Guangzhou, China
Bio-protocol author page: a876
 and Jian-Guo He
Jian-Guo HeAffiliation: School of Marine Sciences, Sun Yat-Sen University, Guangzhou, China
For correspondence: relike2004@sina.com
Bio-protocol author page: a877
Vol 3, Iss 19, 10/5/2013, 4675 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.923

[Abstract] Virus overlay assay is a method to detect protein-protein interaction in vitro. We performed the virus overlay assay to identify the receptor proteins interacting with the infectious spleen and kidney necrosis virus (ISKNV).
Keywords: Far-Western blotting(Far-Western-Blotting), Protein(蛋白质), Virus(病毒)

[Abstract]

Materials and Reagents

  1. Purified ISKNV (was purified in our laboratory)
  2. 12% SDS-PAGE gel
  3. PVDF membrane (Whatman, catalog number: 10485290 )
  4. Coomassie brilliant blue G-250 (Beyotime Institute of Biotechnology, catalog number: P0017B )
  5. Tris-HCl (pH 7.4)
  6. EDTA
  7. Tween 20
  8. Guanidine hydrochloride (Sigma-Aldrich, catalog number: G4505 )
  9. Nonfat milk powder
  10. Maltose-binding protein (MBP) or MBP-mCav-1 protein (these proteins were made in our laboratory)
  11. Anti-MBP antibodies (Sigma-Aldrich, catalog number: M1321 )
  12. Horse radish peroxidase (HRP)-conjugated goat anti-mouse secondary antibodies (Invitrogen, catalog number: 626520 )
  13. HRP substrate solution (Beyotime Institute of Biotechnology, catalog number: P0209 )
  14. BCA protein assay kit (Thermo Fisher Scientific, catalog number: 23225 )
  15. Renaturing buffer (see Recipes)
  16. Tris Buffered Saline and Tween 20 (TBST) (see Recipes)

Equipment

  1. Electrophoresis equipment
  2. Bio-Rad blotting equipment (Bio-Rad Laboratories, model: A101441 )

Procedure

  1. The protein concentrations of the purified virus are determined using the BCA protein assay kit. 100 micrograms of the sample are boiled in 2x SDS-PAGE sample loading buffer for 5 min.
  2. 30 μl (100 micrograms) denatured product is resolved in two parallel 12% SDS-PAGE gels.
  3. After electrophoresis, the viral structural proteins in one gel are transferred to a PVDF membrane by electroblotting using the Bio-Rad wet transfer Blotter (50 V for 2 h), whereas the other gel is stained with Coomassie brilliant blue G-250 for a parallel experiment.
  4. The membrane is washed twice in TBST at room temperature (RT) under constant rotation for 5 min each.
  5. The blots are then blocked overnight in renaturing buffer at 4 °C, and then incubated with 10 μg/ml MBP or MBP-mCav-1 protein for 2 h at RT.
  6. The membrane is incubated with anti-MBP antibodies (1:2,000 dilution) in TBST for 2 h at RT after washing three times in TBST under constant rotation for 10 min each.
  7. The membrane is washed as described in step 4, and the antigen-antibody complexes are then detected using HRP-conjugated goat anti-mouse secondary antibodies (1:5,000 dilution) in TBST for 1 h under constant rotation.
  8. The membrane is washed as described in step 4, the immobilized conjugates on the membrane are subsequently visualized using an HRP substrate solution. When the member reaches desired intensity, pour off the HRP substrate solution and rinse in several changes of distilled water. Incubate in ddH2O for ~20 min, see an example of the results in Figure 1.


    Figure 1. A. Far-Western blot analysis; B. Purified ISKNV particles (100 μg) were isolated using SDS-PAGE, and then stained with Coomassie brilliant blue R-250.

Recipes

  1. Renaturing buffer
    20 mM Tris-HCl
    1 mM EDTA
    0.5 mol/L NaCl
    0.05% Tween 20
    1 M guanidine hydrochloride
    5% nonfat milk powder
  2. TBST (1 L)
    NaCl                8 g
    KCl                 0.2 g
    Tris base         3 g
    Mix in ~ 800 ml dH2O, adjust pH to 7.4 with HCl, then adjust volume to 1 L
    Autoclave
    After cooling, add 1 ml Tween 20

Acknowledgments

This protocol is adapted from Kikkert et al. (1998).

References

  1. Kikkert, M., Meurs, C., van de Wetering, F., Dorfmuller, S., Peters, D., Kormelink, R. and Goldbach, R. (1998). Binding of tomato spotted wilt virus to a 94-kDa thrips protein. Phytopathology 88(1): 63-69.

材料和试剂

  1. 纯化的ISKNV(在我们的实验室中纯化)
  2. 12%SDS-PAGE凝胶
  3. PVDF膜(Whatman,目录号:10485290)
  4. 考马斯亮蓝G-250(Beyotime Institute of Biotechnology,目录号:P0017B)
  5. Tris-HCl(pH 7.4)
  6. EDTA
  7. 吐温20
  8. 盐酸胍(Sigma-Aldrich,目录号:G4505)
  9. 脱脂奶粉
  10. 麦芽糖结合蛋白(MBP)或MBP-mCav-1蛋白(这些蛋白在我们的实验室中制备)
  11. 抗MBP抗体(Sigma-Aldrich,目录号:M1321)
  12. 辣根过氧化物酶(HRP)缀合的山羊抗小鼠第二抗体(Invitrogen,目录号:626520)
  13. HRP底物溶液(Beyotime Institute of Biotechnology,目录号:P0209)
  14. BCA蛋白测定试剂盒(Thermo Fisher Scientific,目录号:23225)
  15. 复性缓冲液(参见配方)
  16. Tris缓冲盐水和Tween 20(TBST)(参见配方)

设备

  1. 电泳设备
  2. Bio-Rad印迹设备(Bio-Rad Laboratories,型号:A101441)

程序

  1. 使用BCA蛋白测定试剂盒测定纯化病毒的蛋白质浓度。 将100微克样品在2×SDS-PAGE样品上样缓冲液中煮沸5分钟。
  2. 将30μl(100微克)变性产物在两个平行的12%SDS-PAGE凝胶中分离。
  3. 电泳后,使用Bio-Rad湿转移Blotter(50V,2小时)通过电印迹将一个凝胶中的病毒结构蛋白转移到PVDF膜,而另一个凝胶用考马斯 亮蓝G-250用于平行实验。
  4. 将膜在室温(RT)下在TBST中在恒定旋转下洗涤两次,每次5分钟。
  5. 然后将印迹在4℃的复性缓冲液中封闭过夜,然后在室温下用10μg/ml MBP或MBP-mCav-1蛋白孵育2小时。
  6. 将膜与TBST中的抗MBP抗体(1:2,000稀释)在室温下孵育2小时,在TBST中在恒定旋转下洗涤三次,每次10分钟。
  7. 如步骤4所述洗涤膜,然后在恒定旋转下使用HRST-缀合的山羊抗小鼠二抗(1:5,000稀释)在TBST中检测抗原 - 抗体复合物1小时。
  8. 如步骤4所述洗涤膜,随后使用HRP底物溶液使膜上的固定的缀合物显色。当成员达到所需强度时,倒出HRP底物溶液并在几次更换的蒸馏水中冲洗。在ddH sub 2 O中孵育约20分钟,参见图1中的结果实例。


    图1。 A。 远Western印迹分析; B.使用SDS-PAGE分离纯化的ISKNV颗粒(100μg),然后用考马斯亮蓝R-250染色。

食谱

  1. 复原缓冲区
    20mM Tris-HCl
    1mM EDTA
    0.5 mol/L NaCl
    0.05%吐温20 1M盐酸胍 5%脱脂奶粉
  2. TBST(1 L)
    NaCl                8 g
    KCl                0.2 g
    Tris碱       3克
    在〜800ml dH 2 O中混合,用HCl调节pH至7.4,然后将体积调节至1L
    高压灭菌器
    冷却后,加入1ml吐温20 /

致谢

该协议改编自Kikkert等人(1998)。

参考文献

  1. Kikkert,M.,Meurs,C.,van de Wetering,F.,Dorfmuller,S.,Peters,D.,Kormelink,R.and Goldbach,R。(1998)。 番茄斑点萎ilt病毒与94-kDa蓟马蛋白的结合。 Phytopathology 88(1):63-69。
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How to cite this protocol: Jia, K., Guo, C., Yang, X. and He, J. (2013). Virus Overlay Assay (Far-Western blotting). Bio-protocol 3(19): e923. DOI: 10.21769/BioProtoc.923; Full Text



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