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RhoGTPase Activation Assay
Rho GTP酶活化试验   

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Abstract

This protocol describes procedures to assay for GTP-bound (active) form of small GTPases of the Rho superfamily in human brain cancer (glioma) cell lines but can also be applied to cells or tissues of other origins. The principle of assay is based on the property of these active GTPases to interact with their specific effectors (e.g. Rhotekin for RhoA; p21-activated kinase (PAK) for Rac1 and Cdc42. In essence, Rhotekin or PAK1 are expressed in the form of GST-fusion protein to pull down the corresponding active GTPases.

Materials and Reagents

  1. Complete Protease Inhibitor Cocktail, EDTA-free (Roche Diagnostics GmbH, catalog number: 11873580001 ), prepared as 25x stock according to manufacturer’s instructions
  2. GST-Rhotekin (Millipore/Upstate, catalog number: 14-662 )
  3. GST-PAK1 (in-house)
  4. anti-RhoA antibody (1:200) (Santa Cruz, catalog number: sc-418 )
  5. anti-Rac1 antibody (1:200) (Santa Cruz, catalog number: sc-95 )
  6. anti-Cdc42 antibody (1:500) (Santa Cruz, catalog number: sc-87 )
  7. anti-GST antibody (1:2,000) (Santa Cruz, catalog number: sc-138 )
  8. Glutathione (GSH) sepharose 4B (GE Healthcare Life Sciences, catalog number: 17-0756-01 )
  9. Dithiothreitol (DTT) (Bio-Rad Laboratories, catalog number: 161-0611 )
  10. Triton X-100 (Bio-Rad Laboratories, catalog number: 161-0407 )
  11. Phosphate-buffered saline (PBS)
  12. Tris-HCl (pH 7.4)
  13. NaCl
  14. EDTA
  15. NP40
  16. Na2P2O7
  17. NaF
  18. Na3VO4
  19. Sample loading buffer
  20. 10% or 12% SDS-PAGE gel
  21. GST pull-down core buffer (see Recipes) 
  22. RIPA buffer (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, catalog number: 5415R )
  2. Nutating mixer (Labnet International)
  3. 1.5 ml Eppendorf tubes
  4. Heat block

Procedure

  1. Lyse cell samples by incubating in RIPA buffer supplemented with 1% Triton X-100 at 4 °C for 2 h. Centrifuge at 18,000 x g for 10 min. at 4 °C and collect the cleared crude cell lysate.
  2. Transfer 15 μl of GSH-sepharose into 100 μl of GST pull-down core buffer in a 1.5 ml Eppendorf tube. Mix well and spin at 835 x g for 1 min. at room temperature. Decant the supernatant and save the washed GSH-sepharose for pre-clearing the samples (step 3).
  3. Pre-clear 100 μg of crude cell lysates by adding into 300 μl of GST pull-down buffer (prepared by supplementing core buffer with 0.1% Triton X-100, 1 mM DTT, and 1x protease inhibitors) containing the washed GSH-sepharose (prepared in step 2). Incubate with agitation at 4 °C for 1 h.
    Note: If multiple samples, first bring up 100 μg lysate to equal volume with corresponding lysis buffer to ensure equal input.
  4. Centrifuge at 835 x g for 1 min at 4 °C. Transfer supernatant to a new 1.5 ml tube without disturbing the sepharose. Save an aliquot of the pre-cleared lysate as “sample input”.
  5. Add to the supernatant 30 μl glutathione beads bound with 30 μg GST-Rhotekin (for RhoA-GTP assay) or GST-PAK1 (for Rac1- and Cdc42-GTP assays). A “GST only” control is similarly prepared by adding GST pre-bound sepharose. Mix well and incubate at 4 °C for 2 to 3 h with rocking.
  6. Centrifuge at 835 x g for 1 min at 4 °C to pellet the sepharose beads.
  7. Aspirate the supernatant (optional: Save supernatant for quality control of pull-down reaction).
  8. Wash the pelleted sepharose beads by adding 300 μl 1x PBS at 4 °C.
  9. Spin at 835 x g for 1 min at 4 °C and aspirate supernatant (Optional: Save supernatant as “first wash” for quality control of washing step).
  10. Repeat washing. Save supernatant as “second wash” (Optional).
  11. Boil the beads in sample loading buffer for 5 min. and spin to harvest the pull-down proteins.
  12. Resolve the eluted proteins and an aliquot of cell/tissue lysates (sample input) by SDS-PAGE (10% or 12% gel). An aliquot of the “supernatant”, “first wash” and “second wash” collected above can also be included for quality control.
  13. Western blot analysis is then performed using anti-RhoA, Rac1, or Cdc42 antibodies to detect active (pull-down) and total (input) GTPases. The blot is also probed with anti-GST antibody to confirm successful GST pull down at comparable efficiency across samples.
    Note: Both Rhotekin and PAK1 preferentially bind to active (GTP-bound form) RhoA and Rac1/Cdc42, respectively. Quality control of these reagents can be performed by incubating GST-Rhotekin (or GST-PAK1) with recombinant RhoA (or Rac1/Cdc42) that has been preloaded with GDP or nonhydrolyzable GTP-γS.

Recipes

  1. GST pull-down core buffer
    20 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1 mM EDTA
  2. RIPA buffer
    50 mM Tris-HCl (pH 7.4)
    150 mM NaCl
    1% NP40
    1 mM Na2P2O7
    1 mM NaF
    1 mM EDTA
    2 mM Na3VO4

Acknowledgments

This protocol is adapted from Li et al. (2012).

References

  1. Li, X., Law, J. W. and Lee, A. Y. (2012). Semaphorin 5A and plexin-B3 regulate human glioma cell motility and morphology through Rac1 and the actin cytoskeleton. Oncogene 31(5): 595-610.

简介

属于黄病毒科的JEV( 病毒)与CLEC5A(C型凝集素结构域家族5,成员A) C型凝集素,与DAP12信号传导蛋白相关并且在骨髓细胞上表达,与登革热病毒的程度相同。 该方案用于通过ELISA方法进行和确定纯化的JEV颗粒与人和鼠CLEC5A.Fc融合蛋白之间的相互作用。 不同的菌株和批次的纯化JEV以及CLEC5A.Fc融合蛋白的纯度可能影响吸光度的结果。 如果用户打算进行ELISA测定,则很可能需要修饰。...

材料和试剂

  1. 完全蛋白酶抑制剂混合物,无EDTA(Roche Diagnostics GmbH,目录号:11873580001),根据制造商的说明书制备为25x母液。
  2. GST-Rhotekin(Millipore/Upstate,目录号:14-662)
  3. GST-PAK1(内部)
  4. 抗-RhoA抗体(1:200)(Santa Cruz,目录号:sc-418)
  5. 抗Rac1抗体(1:200)(Santa Cruz,目录号:sc-95)
  6. 抗Cdc42抗体(1:500)(Santa Cruz,目录号:sc-87)
  7. 抗-GST抗体(1:2,000)(Santa Cruz,目录号:sc-138)
  8. 谷胱甘肽(GSH)琼脂糖4B(GE Healthcare Life Sciences,目录号:17-0756-01)
  9. 二硫苏糖醇(DTT)(Bio-Rad Laboratories,目录号:161-0611)
  10. Triton X-100(Bio-Rad Laboratories,目录号:161-0407)
  11. 磷酸盐缓冲盐水(PBS)
  12. Tris-HCl(pH 7.4)
  13. NaCl
  14. EDTA
  15. NP40
  16. Na 2
  17. NaF
  18. Na 3 VO 4
  19. 样品加载缓冲区
  20. 10%或12%SDS-PAGE凝胶
  21. GST下拉核心缓冲区(见Recipes) 
  22. RIPA缓冲区(参见配方)

设备

  1. 离心机(Eppendorf,目录号:5415R)
  2. 营养搅拌机(Labnet International)
  3. 1.5 ml Eppendorf管
  4. 热块

程序

  1. 通过在补充有1%Triton X-100的RIPA缓冲液中在4℃孵育2小时来裂解细胞样品。 以18,000×g离心10分钟。 在4℃下收集澄清的粗细胞裂解液
  2. 转移15微升GSH琼脂糖凝胶到100微升GST下拉核心缓冲液在1.5毫升离心管中。充分混合并在835×g下旋转1分钟。 。倾析上清液并保存洗涤的GSH-sepharose用于预清洗样品(步骤3)
  3. 通过加入300μlGST下拉缓冲液(通过补充含0.1%Triton X-100,1mM DTT和1×蛋白酶抑制剂的核心缓冲液制备)预清洗100μg粗细胞裂解物,所述缓冲液含有洗涤的GSH-琼脂糖在步骤2)中制备。在4℃下搅拌孵育1小时 注意:如果有多个样品,首先使用相应的裂解缓冲液将100μg裂解液加至等体积以确保相等的输入。
  4. 在4℃下以835×g离心1分钟。转移上清到新的1.5毫升管,而不扰乱琼脂糖。将预澄清的裂解物的等分试样保存为"样品输入"
  5. 添加到上清中与30μgGST-Rhotekin(用于RhoA-GTP测定)或GST-PAK1(用于Rac1-和Cdc42-GTP测定)结合的30μl谷胱甘肽珠。通过添加GST预结合的琼脂糖类似地制备"仅GST"对照。混匀,在4℃下摇动培养2〜3小时。
  6. 在4℃下以835×g离心1分钟以沉淀sepharose珠。
  7. 吸出上清液(可选:保存上清液以进行下拉反应的质量控制)。
  8. 通过在4℃下加入300μl1×PBS洗涤沉淀的琼脂糖凝胶珠子
  9. 在4℃下以835×g离心1分钟并抽吸上清液(可选:将上清液保存为"第一次洗涤",用于洗涤步骤的质量控制)。
  10. 重复洗涤。 将上清液保存为"第二次洗涤"(可选)。
  11. 将样品加样缓冲液中的珠子煮沸5分钟。 并旋转以收获下拉蛋白
  12. 通过SDS-PAGE(10%或12%凝胶)分离洗脱的蛋白质和一等份的细胞/组织裂解物(样品输入)。 还可以包括上面收集的"上清液","第一次洗涤"和"第二次洗涤"的等分试样以进行质量控制。
  13. 然后使用抗RhoA,Rac1或Cdc42抗体进行Western印迹分析以检测活性(下拉)和总(输入)GTP酶。 还用抗GST抗体探测印迹以确认成功的GST下拉,在样品之间具有相当的效率 注意:Rhotekin和PAK1优先分别结合活性(GTP结合形式)RhoA和Rac1/Cdc42。 这些试剂的质量控制可以通过将GST-Rhotekin(或GST-PAK1)与预装有GDP或不可水解的GTP-γS的重组RhoA(或Rac1/Cdc42)孵育来进行。

食谱

  1. GST下拉内核缓冲区
    20mM Tris-HCl(pH7.4) 150mM NaCl 1mM EDTA
  2. RIPA缓冲区
    50mM Tris-HCl(pH7.4) 150mM NaCl 1%NP40
    1mM Na 2 P 2 O 7 sub
    1 mM NaF
    1mM EDTA
    2mM Na 3+ VO 4

致谢

该协议改编自Li等人(2012)。

参考文献

  1. Li,X.,Law,J.W.and Lee,A.Y。(2012)。 Semaphorin 5A和神经丛素-B3通过Rac1和肌动蛋白细胞骨架调节人类神经胶质瘤细胞运动和形态。/a> Oncogene 31(5):595-610。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lee, A. Y. (2012). RhoGTPase Activation Assay. Bio-protocol 2(19): e269. DOI: 10.21769/BioProtoc.269.
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