Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed

RAB21 Activity Assay Using GST-fused APPL1

使用GST融合 APPL1进行RAB21活性试验

Steve Jean Steve Jean
Amy A. Kiger Amy A. Kiger

发布: 2016年02月20日第6卷第4期 DOI: 10.21769/BioProtoc.1738 浏览次数: 8152

评审: Ralph BottcherVaibhav B ShahAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of EMBO Reports, featuring study using the protocol.

Mar 2015

Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

96孔板NF-κB/AP-1报告基因检测转染细胞中野生型及致病性ALPK1突变体活性

96孔板NF-κB/AP-1报告基因检测转染细胞中野生型及致病性ALPK1突变体活性

Tom Snelling

2024年11月20日 983 阅读

野生型和致病突变体ALPK1激酶活性的无细胞放射性磷酸化定量测定

野生型和致病突变体ALPK1激酶活性的无细胞放射性磷酸化定量测定

Tom Snelling

2024年11月20日 943 阅读

基于荧光偏振的组织蛋白酶L抑制剂高通量筛选方法

基于荧光偏振的组织蛋白酶L抑制剂高通量筛选方法

Keyu Guo [...] Shuyi Si

2025年07月20日 1236 阅读

Abstract

The Rab family of small GTPases are essential regulators of membrane trafficking events. As with other small GTPase families, Rab GTPases cycle between an inactive GDP- bound state and an active GTP-bound state. Guanine nucleotide exchange factors (GEFs) promote Rab activation with the exchange of bound GDP for GTP, while GTPase-activating proteins (GAPs) regulate Rab inactivation with GTP hydrolysis. Numerous methods have been established to monitor the activation status of Rab GTPases. Of those, FRET-based methods are used to identify when and where a Rab GTPase is activated in cells. Unfortunately, the generation of such probes is complex, and only a limited number of Rabs have been probed this way. Biochemical purification of activated Rabs from cell or tissue extracts is easily achievable through the use of a known Rab effector domain to pull down a specific GTP-bound Rab form. Although this method is not ideal for detailed subcellular localization, it can offer temporal resolution of Rab activity. The identification of a growing number of specific effectors now allows tests for activation levels of many Rab GTPases in specific conditions. Here, we described an affinity purification approach using GST fused APPL1 (a known RAB21 effector) to test RAB21 activation in mammalian cells. This method was successfully used to assay changes in RAB21 activation status under nutrient rich versus starved conditions and to test the requirement of the MTMR13 RAB21 GEF in this process.

Keywords: Membrane trafficking (膜转运) search Rab GTPase (Rab GTPase) search Autophagy (自噬) search

Materials and Reagents

  1. 50 ml conical tube (Sarstedt AG & CO, catalog number: 62.547.004 )
  2. 100 mm Tissue Culture Dish (Corning, catalog number: 430167 )
  3. 0.22 µm filtering unit (Genesee Scientific Corporation, catalog number: 25-227 ) 

  4. Cell lifter (Corning, catalog number: 3008 )
  5. Escherichia coli BL21 (New England Biolabs, catalog number: C2530H ) 

  6. pGEX-5X-3 (GE Healthcare, catalog number: 27-4586-01 )
  7. pGEX5X3-Happl1 (aa 5-419) (self-made) (Jean et al., 2015)
  8. pAcEGFP-C1 vector (Clontech, catalog number: 632470 )
  9. pEGFP-C1:RAB21 wild type [human RAB21 cloned in pAcEGFP-C1 (this construct was used to generate the stable HeLa M cell line)] (self-made) (Jean et al., 2015)
  10. EGFP:RAB21wt (wildtype) stably-transfected HeLa M cells (self-made) (Jean et al., 2015) 

  11. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Biopioneer, catalog number: c0012 ) 

  12. Ampicillin Sodium Salt (Crystalline Powder) (Thermo Fisher Scientific, catalog number: BP176025 ) 

  13. BD BactoTM Tryptone (Thermo Fisher Scientific, catalog number: DF0123173 ) 

  14. Yeast Extract (Thermo Fisher Scientific, catalog number: 212750 ) 

  15. Glutathione Sepharose 4B beads (GE Healthcare, catalog number: 17-0756-01 ) 

  16. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: P8340-5 ) 

  17. Fetal Bovine Serum (Sigma-Aldrich, catalog number: F2442-500 ml ) 

  18. Penicillin-Streptomycin solution (Life Technologies, catalog number: 15140-122 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  19. Trypsin-EDTA (0.25%), phenol red (Life Technologies, catalog number: 25200-056 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25200-056”.
  20. 0.4% Trypan-Blue (Life Technologies, InvitrogenTM, catalog number: 15250-061 ) 

    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15250-061”.
  21. Guanosine 5’-triphosphate sodium salt hydrate (Sigma-Aldrich, catalog number: G8877-25 mg ) 

  22. 0.5 M liquid EDTA (Cell grow, catalog number: 45001-122 ) 

    Note: Currently, it is “VWR International, catalog number: 45001-122 ”.
  23. Earl’s Balanced Salt Solution (with sodium bicarbonate, without phenol red liquid, sterile-filtered) (Sigma-Aldrich, catalog number: E3024-500 ml ) 

  24. Dithiothreitol (VWR International, catalog number: IB21040 ) 

  25. HEPES (Thermo Fisher Scientific, Biotech, catalog number: BP310-100 ) 

  26. IGEPAL CA-630 (Sigma-Aldrich, catalog number: I8896-100 ml ) 

  27. Glycerol (Thermo Fisher Scientific, catalog number: BP229-1 ) 

  28. Magnesium Chloride Hexahydrate (MgCl2.6H2O) (Thermo Fisher Scientific, catalog number: BP214-500 ) 

  29. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: S6508-10 g ) 

  30. Triton X-100 (Sigma-Aldrich, catalog number: X100-500 ml ) 

  31. Sodium Chloride (NaCl) (Thermo Fisher Scientific, catalog number: S671-3 ) 

  32. Potassium Chloride (KCl) (VWR International, catalog number: BDH9258-500 g ) 

  33. Sodium Phosphate Dibasic Anhydrous (Na2HPO4) (Thermo Fisher Scientific, catalog number: S374500 ) 

  34. Potassium Phosphate Monobasic (KH2PO4) (Thermo Fisher Scientific, catalog number: P285-S500 ) 

  35. Dubelcco Modified Eagle Medium with High glucose with 4 mM L-Glutamine and sodium pyruvate (GE Healthcare, HycloneTM, catalog number: SH30243.FS ) (see Recipes for complete DMEM) 

  36. Anti-GFP (B2) antibody (Santa Cruz Biotechnology, catalog number: sc-9996 )
  37. Luria Broth (see Recipes) 

  38. LB/ampicillin (see Recipes) 

  39. Complete DMEM (see Recipes) 

  40. 1x Phosphate Buffer Saline (PBS) (see Recipes)
  41. 1 M HEPES (pH 7.5) (see Recipes) 

  42. 5 M NaCl (see Recipes)
  43. 1 M MgCl2 (see Recipes)
  44. 0.2 M Sodium Orthovanadate (see Recipes)
  45. 25 mM GTP (see Recipes) 

  46. Modified Magnesium Lysis Buffer (MLB) (see Recipes)

Equipment

  1. Pipettes 10 μl, 100 μl and 1,000 μl (Eppendorf, catalog numbers: 3120000020 , 3120000046 and 31200000623 )
  2. Sonifier® cell disrupters (VWR International, catalog number: CA33995-320 )
  3. 37 °C shaker [similar to New Brunswick Excella E25 (Eppendorf, New Brunswick Scientific, catalog number: M1353-0002 )] 

  4. 30 °C shaker [similar to New Brunswick Excella E25 (Eppendorf, New Brunswick Scientific, catalog number: M1353-0002 )]

  5. Tissue culture biosafety cabinet [similar to Thermo Scientific 1300 Series Class II (Thermo Fisher Scientific, catalog number: 1323 )]

  6. Hemacytometer (VWR International, catalog number: 100498-470 ) 

  7. Centrifuge (Eppendorf, catalog number: 5804R ) 

  8. Table top centrifuge (Eppendorf, catalog number: 5415D ) 

  9. Rotating wheel (Barnstead Thermolyne Lab Quake Shaker Rotisserie) 

  10. Sonicator, Branson sonifier S250A Analog Ultrasonic Cell Disruptor (Thermo Fisher Scientific, catalog number: 22309782 )

Procedure

English
中文翻译

文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Jean, S. and Kiger, A. A. (2016). RAB21 Activity Assay Using GST-fused APPL1. Bio-protocol 6(4): e1738. DOI: 10.21769/BioProtoc.1738.
  2. Jean, S., Cox, S., Nassari, S. and Kiger, A. A. (2015). Starvation-induced MTMR13 and RAB21 activity regulates VAMP8 to promote autophagosome-lysosome fusion. EMBO Rep 16(3): 297-311.

    3. ris ris Download Citation in RIS Format

分类

生物化学 > 蛋白质 > 活性

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

0/150

tip 提问指南

+ 问题描述

写下详细的问题描述，包括所有有助于他人回答您问题的信息（例如实验过程、条件和相关图像等）。

post 发布问题
0 Q&A
pen 提交稿件