搜索

RAB21 Activity Assay Using GST-fused APPL1
使用GST融合 APPL1进行RAB21活性试验   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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), Rab GTPase(Rab GTPase), Autophagy(自噬)

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

  1. GST:APPL1 (adaptor protein containing PH domain, PTB domain and Leucine zipper motif) bacterial expression
    1. Using sterile technique, strike an LB-ampicillin petri dish of E. coli BL21 transformed with pGEX5X3-hAPPL1 (aa 5-419), and incubate at 37 °C overnight (o/n). Plates should be used within 1 
week to have maximal protein expression. 

    2. Inoculate 100 ml of LB/ampicillin with one colony from the stroke plate. Grow under constant 
agitation at 37 °C o/n. 

    3. Centrifuge the o/n culture at 4,000 x g for 10 min. 

    4. Resuspend the pellet in 50 ml of LB/ampicillin, and mix with 950 ml of LB/ampicillin. 

    5. Grow the culture at 30 °C with agitation until an OD600 of 0.8 is reached, usually by 90 min, then induce the expression of GST:hAPPL1 (5-419) by adding a final concentration of 1 mM 
IPTG. Incubate for 3 h at 30 °C under constant agitation. 

    6. After induction, pellet the cells by centrifugation at 4,000 x g for 10 min at 4 °C. Wash the pellet 
once with 50 ml of 1x PBS. Pellet the cells again by centrifugation. Remove the PBS, and freeze the pellet at -80 °C or move on with the purification. 


  2. GST:APPL1 (5-419) purification
    1. Resuspend bacteria in 25 ml ice-cold 1x PBS containing 1x Protease inhibitor cocktail and 1 mM DTT, and transfer the cell suspension into a 50 ml conical tube. 

    2. Sonicate four times for 45 sec each time at 100% duty cycle and at an output of 4 (using a Branson sonifier). Make sure to cool down the sample between each sonication. 

    3. Add 2.5 ml of a 10% Triton X-100/PBS solution to the bacterial lysate for a final Triton concentration of 1%. 

    4. Sonicate as above 1 more time. 

    5. To pellet bacterial debris, centrifuge for 20 min at 12,000 x g at 4 °C. 

    6. While the centrifugation is ongoing, prepare Glutathione Sepharose 4B beads:
      1. Pipet 800 μl of glutathione beads into an Eppendorf tube. 

      2. Centrifuge beads in a table top centrifuge for 30 sec at 12,000 x g at room 
temperature.
      3. Remove supernatant, and add 1 ml of 1x sterile PBS. Resuspend Glutathione Sepharose 4B beads. 

      4. Repeat steps B6b-c for two more times. 

      5. After the last wash, resuspend Glutathione Sepharose 4B beads in 500 μl of 1x PBS.
      6. Transfer the resuspended Glutathione Sepharose 4B beads into a 50 ml conical tube.

    7. After centrifugation of the bacterial lysate, pour the supernatant into the 50 ml conical tube containing the prepared Glutathione Sepharose 4B beads. Avoid transferring any E. coli crude. 

    8. To avoid degradation of GST:APPL1 during the room temperature incubation, add 250 μl of 100x Protease inhibitor cocktail to the slurry. 

    9. Incubate with slight agitation 20 min at room temperature. 

    10. Centrifuge the slurry in a tabletop centrifuge at 4,000 x g for 3 min at 4 °C. 

    11. Remove the supernatant and resuspend Glutathione Sepharose 4B beads in 45 ml of 1x ice-cold PBS. 

    12. Repeat steps B10-11 four more times. 

    13. After the last wash, remove most of the 1x PBS. Transfer the slurry to an Eppendorf tube, and assess the quality and quantity of the purified protein through regular SDS-PAGE electrophoresis followed by Coomassie staining (Figure 1). We suggest to use increasing amounts of the GST:APPL1 slurry to assess purity (1 µl, 5 µl, 10 µl and 20 µl of the slurry will cover a large range of protein concentration). Serial BSA dilution series can be used for quantification. 

    14. Store beads at 4 °C. GST:APPL1 beads were used in the same month after their preparation, and experiments were successfully reeated over a month. However, we haven't tested longer storage time.


      Figure 1. Coomasie stained PVDF membrane of purified GST:APPL1

  3. GFP:RAB21wt HeLa cell culture and autophagy induction
    1. Remove media from 100 mm stock GFP:RAB21wt cell culture plate, and wash with 5 ml of 1x PBS. 

    2. Remove PBS, add 1 ml of 0.25% trypsin-EDTA, then incubate at 37 °C for 3 min. 

    3. Wash cells off the plate with 10 ml of complete DMEM. 

    4. Pipet 20 μl of the cell suspension, and add to 100 μl of Trypan-Blue solution. 

    5. Pipet 10 μl of this cell/Trypan-Blue dilution into a hemacytometer chamber. 

    6. Count viable cells using the hemacytometer on a standard microscope. Viable cells will be Trypan-Blue negative. 

    7. Add a total of 5 x 105 cells per 100 mm dish (1 dish is required per time point). 

    8. Bring to 8 ml per dish with complete DMEM, and move to 37 °C incubator overnight (o/n). 

    9. To elicit starvation-induced autophagy, wash the cells 1x with 5 ml of pre-warmed EBSS. 

    10. Remove the EBSS, and add 8 ml of pre-warmed EBSS. Incubate for the desired amount of time (typical time points for GFP:Rab21wt HeLa cells, were 0, 5, 15, 30 and 60 min) at 37 °C. To ease subsequent manipulations, start with the longest time points first so that all the different 
timepoints can be processed concurrently. 


  4. RAB21 activity assay
    1. Transfer cells on ice, and wash cells twice with ice cold 1x PBS. 

    2. Remove PBS, and lyse cells in 800 μl of ice-cold modified MLB buffer. Scrape cells off the
plate using a cell lifter, and transfer cell lysate to a 1.5 ml Eppendorf tube. 

    3. Incubate cell lysate for 20 min on ice. 

    4. Centrifuge cell lysate 10 min at 16,100 x g at 4 °C. 

    5. While centrifugation is in progress, prepare the GST:APPL1 beads for the experiment:
      1. Resuspend GST:APPL1 beads, and pipet the appropriate volume into a new 1.5 ml Eppendorf tube. 4 μg of GST:APPL1 is required per time point, thus if 5 time points are tested and the GST:APPL1 is at a concentration of 160 ng/μl, then 25 μl of GST:APPL1 is required per tube. 

      2. Centrifuge the GST:APPL1 beads 30 sec at 11,000 x g at 4 °C. 

      3. Remove supernatant, and add 1 ml of modified MLB buffer. 

      4. Repeat steps D5b-c two more times. 

      5. Remove supernatant. 

    6. For each time point, add 700 μl of the GFP:RAB21wt HeLa cell supernatant to the prepared beads. If different cell lines are compared, the total amount of protein for each cell line lysate must be measured and adjusted in order to have equal amounts of protein incubated with GST:APPL1.
    7. Incubate on a rotating wheel for 1 h at 4 °C. 

    8. Centrifuge each tube 30 sec at 11,000 x g at room temperature 

    9. Remove supernatants, and add 1 ml of ice-cold modified MLB buffer. 

    10. Repeat steps 8 and 9 three times (for a total of four washes). 

    11. Elute bound proteins from beads by adding 30 μl of 2x SDS-PAGE loading buffer, and heat 
at 95 °C for 5 min. 

    12. Analyze the result through western blot analysis with antibodies against GFP (Figure 2).


      Figure 2. Typical result of a GFP:RAB21 activity assay. Upon nutrient deprivation, GFP:RAB21 is transiently activated. GFP:RAB21-GTP was detected by GST:APPL1 pull-down. Total RAB21 represents RAB21 in cell lysates. Both RAB21-total and RAB21-GTP were detected with an anti-GFP western blot.


Recipes

  1. Luria Broth (LB)

    Mix 10 g of Tryptone, 5 g of yeast extract and 10 g of NaCl
    Add 1 L of water
    Mix well and autoclave for 30 min at 121 °C
  2. LB/ampicillin

    To 1 L of LB, add 1 ml of ampicillin stock solution (50 mg/ml)
  3. Complete DMEM

    Remove 55 ml of DMEM from the purchased 500 ml HyClone bottle
    Add 50 ml of fetal bovine serum
    Add 5 ml of Penicillin/Streptomycin solution
    Filter sterilize using filtering device
  4. 1x phosphate buffer saline
(PBS)
    Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4
    Add 900 ml of dH2O
    Adjust pH to 7.4
    Adjust final volume to 1,000 ml

    Sterilized by autoclaving at 121 °C for 20 min
  5. 1 M HEPES (pH 7.5)
    Mix 238.3 g of HEPES in 900 ml of water
    Adjust pH to 7.5 with NaOH
    Adjust to 1,000 ml
    Sterilized by autoclaving at 121 °C for 20 min
  6. 5 M NaCl

    Dissolve 292 g of NaCl in 800 ml of water
    Adjust to a final volume of 1,000 ml
    Sterilized by autoclaving at 121 °C for 20 min
  7. 1 M MgCl2

    Dissolve 203 g of MgCl2.6H2O in 700 ml of water
    Adjust to a final volume of 1,000 ml
    Sterilized by autoclaving at 121 °C for 20 min
  8. 0.2 M sodium orthovanadate

    Dissolve 3.68 g of Na3VO4 in 100 ml of water
    Vortex and aliquot for storage
    Stored at -20 °C
  9. 25 mM GTP
    Add 0.13 g of GTP to 10 ml of water
    Filter using a 0.2 μm filter and aliquot for storage
    Stored at -20 °C
  10. Modified MLB buffer

    Add to 50 ml of water
    2.5 ml of 1 M HEPES (pH 7.5) stock solution
    3 ml of 5 M NaCl stock solution

    2 ml of 1 M MgCl2
    10 ml of glycerol

    1 ml of IGEPAL CA-630
    0.2 ml of 0.5 M EDTA

    0.5 ml of 0.2 M Na3VO4
    0.1 ml of 25 mM GTP stock solution (to maintain RAB21 in its activated state)
    Stored at -20 °C

Acknowledgments

The RAB21 activity assay was adapted from previously published studies (Franco et al., 2014; Mai et al., 2011; Zhu et al., 2007) and was performed in (Jean et al., 2015). This work was supported by FRSQ, AHA and CRS postdoctoral fellowships to SJ, and NIH RO1 GM078176 and support from the SDCSB NIH P50 GM085764 to AAK.

References

  1. Franco, I., Gulluni, F., Campa, C. C., Costa, C., Margaria, J. P., Ciraolo, E., Martini, M., Monteyne, D., De Luca, E., Germena, G., Posor, Y., Maffucci, T., Marengo, S., Haucke, V., Falasca, M., Perez-Morga, D., Boletta, A., Merlo, G. R. and Hirsch, E. (2014). PI3K class II alpha controls spatially restricted endosomal PtdIns3P and Rab11 activation to promote primary cilium function. Dev Cell 28(6): 647-658.
  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. Mai, A., Veltel, S., Pellinen, T., Padzik, A., Coffey, E., Marjomaki, V. and Ivaska, J. (2011). Competitive binding of Rab21 and p120RasGAP to integrins regulates receptor traffic and migration. J Cell Biol 194(2): 291-306.
  4. Zhu, G., Chen, J., Liu, J., Brunzelle, J. S., Huang, B., Wakeham, N., Terzyan, S., Li, X., Rao, Z., Li, G. and Zhang, X. C. (2007). Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5. EMBO J 26(14): 3484-3493.

简介

小GTP酶的Rab家族是膜运输事件的必要调节剂。与其他小GTP酶家族一样,Rab GTP酶在不活动的GDP结合状态和活性GTP结合状态之间循环。鸟嘌呤核苷酸交换因子(GEF)促进Rab激活与交换绑定GDP GTP,而GTPase激活蛋白(GAP)调节Rab失活与GTP水解。已经建立了许多方法来监测Rab GTPases的活化状态。其中,基于FRET的方法用于鉴定细胞中Rab GTPase在何时和何地被激活。不幸的是,这种探针的产生是复杂的,并且只有有限数量的Rab已经以这种方式探测。来自细胞或组织提取物的活化的Rabs的生物化学纯化通过使用已知的Rab效应结构域以下拉特定的GTP结合的Rab形式是容易实现的。虽然这种方法不是理想的详细的亚细胞定位,它可以提供Rab活动的时间分辨率。越来越多的特异性效应物的鉴定现在允许在特定条件下测试许多Rab GTP酶的活化水平。在这里,我们描述了一种亲和纯化方法使用GST融合APPL1(一种已知的RAB21效应)来测试哺乳动物细胞中的RAB21激活。该方法成功地用于测定RAB21激活状态下营养丰富与饥饿条件下的变化,并测试在此过程中MTMR13 RAB21 GEF的需求。

关键字:膜转运, Rab GTPase, 自噬

材料和试剂

  1. 50ml锥形管(Sarstedt AG& CO,目录号:62.547.004)
  2. 100mm组织培养皿(Corning,目录号:430167)
  3. 0.22μm过滤单元(Genesee Scientific Corporation,目录号:25-227)
  4. 电池升降器(Corning,目录号:3008)
  5. 大肠杆菌BL21(New England Biolabs,目录号:C2530H)
  6. pGEX-5X-3(GE Healthcare,目录号:27-4586-01)
  7. pGEX5X3-Happl1(aa 5-419)(自制)(Jean 等人,2015)
  8. pAcEGFP-C1载体(Clontech,目录号:632470)
  9. pEGFP-C1:RAB21野生型[克隆在pAcEGFP-C1中的人类RAB21(此构建体用于产生稳定的HeLa M细胞系)](自制)(Jean等人,2015) br />
  10. EGFP:RAB21wt(野生型)稳定转染的HeLa M细胞(自制)(Jean 等,2015)
  11. 异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)(Biopioneer,目录号:c0012)
  12. 氨苄青霉素钠盐(结晶粉末)(Thermo Fisher Scientific,目录号:BP176025)
  13. BD Bacto TM 胰蛋白胨(Thermo Fisher Scientific,目录号:DF0123173)
  14. 酵母提取物(Thermo Fisher Scientific,目录号:212750)
  15. 谷胱甘肽琼脂糖4B珠(GE Healthcare,目录号:17-0756-01)
  16. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:P8340-5)
  17. 胎牛血清(Sigma-Aldrich,目录号:F2442-500ml)
  18. 青霉素 - 链霉素溶液(Life Technologies,目录号:15140-122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"
  19. 胰蛋白酶-EDTA(0.25%),酚红(Life Technologies,目录号:25200-056) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25200-056"
  20. 0.4%台盼蓝(Life Technologies,Invitrogen TM ,目录号:15250-061)
    注意:目前,"赛默飞世尔科技,Gibco TM <目录号:15250-061"
  21. 鸟苷5'-三磷酸钠盐水合物(Sigma-Aldrich,目录号:G8877-25mg)
  22. 0.5M液体EDTA(Cell growth,目录号:45001-122)
    注意:目前,它是"VWR国际,目录号:45001-122"。
  23. Earl's平衡盐溶液(用碳酸氢钠,不含酚红液,无菌过滤)(Sigma-Aldrich,目录号:E3024-500ml)
  24. 二硫苏糖醇(VWR International,目录号:IB21040)
  25. HEPES(Thermo Fisher Scientific,Biotech,目录号:BP310-100)
  26. IGEPAL CA-630(Sigma-Aldrich,目录号:I8896-100ml)
  27. 甘油(Thermo Fisher Scientific,目录号:BP229-1)
  28. 氯化镁六水合物(MgCl 2·6H 2 O·6H 2 O)(Thermo Fisher Scientific,目录号:BP214-500)
  29. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:S6508-10g)
  30. Triton X-100(Sigma-Aldrich,目录号:X100-500ml)
  31. 氯化钠(NaCl)(Thermo Fisher Scientific,目录号:S671-3)
  32. 氯化钾(KCl)(VWR International,目录号:BDH9258-500g)
  33. 磷酸氢二钠无水(Na 2 HPO 4)(Thermo Fisher Scientific,目录号:S374500)
  34. 磷酸二氢钾(KH 2 PO 4)(Thermo Fisher Scientific,目录号:P285-S500)
  35. 具有高葡萄糖与4mM L-谷氨酰胺和丙酮酸钠的Dubelcco改良Eagle培养基(GE Healthcare,Hyclone TM ,目录号:SH30243.FS)(参见完全DMEM的配方)
  36. 抗GFP(B2)抗体(Santa Cruz Biotechnology,目录号:sc-9996)
  37. Luria Broth(请参阅食谱)
  38. LB /氨苄青霉素(见配方)
  39. 完成DMEM(参见配方)
  40. 1x磷酸盐缓冲盐水(PBS)(参见配方)
  41. 1 M HEPES(pH 7.5)(参见配方)
  42. 5 M NaCl(见配方)
  43. 1 M MgCl <2> (参见配方)
  44. 0.2 M原钒酸钠(参见配方)
  45. 25 mM GTP(参见配方)
  46. 改性镁裂解缓冲液(MLB)(参见配方)

设备

  1. 移液管10μl,100μl和1000μl(Eppendorf,目录号:3120000020,312000046和31200000623)
  2. Sonifier ?细胞破坏剂(VWR International,目录号:CA33995-320)
  3. 37℃摇床[类似于New Brunswick Excella E25(Eppendorf,New Brunswick Scientific,目录号:M1353-0002)]
  4. 30℃摇床[类似于New Brunswick Excella E25(Eppendorf,New Brunswick Scientific,目录号:M1353-0002)]
  5. 组织培养生物安全柜[类似于Thermo Scientific 1300 Series Class II(Thermo Fisher Scientific,目录号:1323)]
  6. 血细胞计数器(VWR International,目录号:100498-470)
  7. 离心机(Eppendorf,目录号:5804R)
  8. 台式离心机(Eppendorf,目录号:5415D)
  9. 旋转轮(Barnstead Thermolyne Lab Quake Shaker Rotisserie)
  10. 超声波仪,Branson超声波仪S250A模拟超声波细胞破碎仪(Thermo Fisher Scientific,目录号:22309782)

程序

  1. GST:APPL1(含有PH结构域,PTB结构域和亮氨酸拉链基序的衔接蛋白)细菌表达
    1. 使用无菌技术,敲击E的LB-氨苄青霉素培养皿。用pGEX5X3-hAPPL1(aa 5-419)转化的大肠杆菌 BL21 ,并在37℃ 过夜(o/n)。板应在1周内使用最大 蛋白表达。
    2. 从中风板接种一个菌落的100毫升LB /氨苄青霉素。在37℃o/n下连续搅拌下生长。
    3. 以4,000×g离心O/n培养物10分钟。
    4. 将沉淀重悬在50ml LB /氨苄青霉素中,并与950ml LB /氨苄青霉素混合。
    5. 在30℃下在搅拌下培养培养物直到OD 600为0.8 达到,通常90分钟,然后诱导表达的GST:hAPPL1 (5-419),通过加入终浓度为1mM IPTG。孵育3小时 在30℃下在恒定搅拌下。
    6. 诱导后, 通过在4℃以4,000×g离心10分钟来分离细胞。洗涤沉淀 ?一次用50ml的1x PBS。通过离心再次沉淀细胞。 取出PBS,并在-80°C冻结沉淀或继续前进 纯化。

  2. GST:APPL1(5-419)纯化
    1. 重悬细菌在25毫升冰冷的1×PBS含有1×蛋白酶 抑制剂混合物和1mM DTT,并将细胞悬浮液转移至 ?50ml锥形管。
    2. 超声处理四次,每次45秒 在100%占空比和在输出4(使用Branson超声波仪)。 确保在每次超声处理之间冷却样品。
    3. 向细菌裂解物中加入2.5ml 10%Triton X-100/PBS溶液,最终Triton浓度为1%。
    4. 超声处理1次以上。
    5. 为了沉淀细菌碎片,在4℃下以12,000xg离心20分钟。
    6. 在离心过程中,准备谷胱甘肽琼脂糖4B珠:
      1. 吸取800微升谷胱甘肽珠进入离心管。
      2. 在台式离心机中在室温下以12,000xg离心珠离心30秒。
      3. 取出上清液,加入1 ml 1×无菌PBS。重悬谷胱甘肽Sepharose 4B珠。
      4. 重复步骤B6b-c两次。
      5. 最后一次洗涤后,重悬在500μl1×PBS中的谷胱甘肽琼脂糖4B珠。
      6. 将重悬的谷胱甘肽Sepharose 4B珠转移到50ml锥形管中。
    7. 离心细菌裂解液后,倒入上清液 进入含有制备的谷胱甘肽的50ml锥形管中 Sepharose 4B珠。避免转移任何 E。大肠杆菌粗品。
    8. 至 避免GST降解:APPL1在室温孵育过程中, 向浆液中加入250μl100x蛋白酶抑制剂混合物。
    9. 在室温下轻微搅拌孵育20分钟。
    10. 在4℃下在台式离心机中以4,000xg离心浆液3分钟。
    11. 取出上清液,并重悬于Glutathione Sepharose 4B珠在45毫升1×冰冷PBS中。
    12. 重复步骤B10-11四次。
    13. 最后一次洗涤后,取出大部分1x PBS。转移浆料 ?到Eppendorf管,并评估其质量和数量 纯化的蛋白通过常规SDS-PAGE电泳,随后 考马斯染色(图1)。我们建议使用越来越多的 GST:APPL1浆液以评估纯度(1μl,5μl,10μl和20μl 该浆料将覆盖大范围的蛋白质浓度)。串行 BSA稀释系列可用于定量。
    14. 商店 珠子在4℃。 GST:APPL1珠在其使用后的同一个月 制备和实验成功地重复了一个月。 但是,我们没有测试更长的存储时间

      图1.纯化的GST的Coomasie染色的PVDF膜:APPL1

  3. GFP:RAB21wt HeLa细胞培养和自噬诱导
    1. 从100mm股票GFP:RAB21wt细胞培养板中删除媒体,并用5ml的1×PBS洗涤。
    2. 取出PBS,加入1ml 0.25%胰蛋白酶-EDTA,然后在37℃孵育3分钟。
    3. 用10ml完全DMEM洗涤板上的细胞。
    4. 吸取20微升的细胞悬液,并加入100微升的台盼蓝溶液。
    5. 吸取10微升的这个单元格/台盼蓝稀释到血细胞计数室。
    6. 在标准显微镜上使用血细胞计数器计数活细胞。活细胞将是台盼蓝阴性。
    7. 每100mm培养皿中加入总共5×10 5个细胞(每个时间点需要1个培养皿)。
    8. 用完全DMEM使每个培养皿达到8ml,并移至37℃培养箱过夜(o/n)。
    9. 为了引起饥饿诱导的自噬,用5毫升预热的EBSS洗涤细胞1×。
    10. 取出EBSS,加入8 ml预热的EBSS。孵化为 期望的时间量(GFP:Rab21wt HeLa细胞的典型时间点, 为0,5,15,30和60分钟)。缓解后续 操作,首先从最长的时间点开始,使所有的 可以同时处理不同的时间点。

  4. RAB21活性测定
    1. 转移细胞在冰上,用冰冷的1×PBS洗涤细胞两次。
    2. 除去PBS,并溶解细胞在800μl冰冷的修改MLB缓冲区。 ?使用细胞提升器和转移细胞从细胞板刮下细胞 裂解物至1.5ml Eppendorf管。
    3. 孵育细胞裂解物在冰上20分钟。
    4. 在4℃下以16,100×g离心细胞裂解物10分钟。
    5. 在离心过程中,准备用于实验的GST:APPL1珠子:
      1. 重悬GST:APPL1珠,并移取适当的体积进入 新的1.5ml Eppendorf管。 4μgGST:每个时间点需要APPL1, ?因此如果测试5个时间点并且GST:APPL1在a 浓度为160ng /μl,则需要25μl的GST:APPL1 管。
      2. 在4℃下,以11,000×g离心GST:APPL1珠30秒。
      3. 除去上清液,加入1ml改性MLB缓冲液。
      4. 重复步骤D5b-c两次。
      5. 除去上清液。
    6. 对于每个时间点,添加700微升GFP:RAB21wt HeLa细胞 上清液至制备的珠。如果比较不同的细胞系, ?必须测量每种细胞系裂解物的蛋白质总量 并调节以使等量的蛋白质孵育 GST:APPL1。
    7. 在4℃下在旋转轮上孵育1小时。
    8. 每个管在室温下以11,000×g离心30秒
    9. 取出上清液,加入1ml冰冷的改性MLB缓冲液。
    10. 重复步骤8和9三次(总共四次洗涤)。
    11. 通过加入30μl的2x SDS-PAGE上样缓冲液从珠子洗脱结合的蛋白质,并在95℃加热5分钟。
    12. 通过使用针对GFP的抗体的western印迹分析来分析结果(图2)。


      图2.GFP:RAB21活性测定的典型结果 剥夺,GFP:RAB21被瞬时激活。 GFP:RAB21-GTP 通过GST:APPL1下拉检测。总RAB21代表细胞中的RAB21 裂解物。用抗GFP检测RAB21总和和RAB21-GTP 免疫印迹。

食谱

  1. Luria肉汤(LB)
    混合10克胰蛋白胨,5克酵母提取物和10克NaCl
    加入1升水
    充分混合并在121℃高压灭菌30分钟
  2. LB /氨苄青霉素
    向1L LB中加入1ml氨苄青霉素储备液(50mg/ml)
  3. 完成DMEM
    从购买的500 ml HyClone瓶中取出55 ml DMEM
    加入50ml胎牛血清
    加入5ml青霉素/链霉素溶液
    使用过滤装置过滤灭菌
  4. 1×磷酸盐缓冲盐水(PBS)
    将8g的NaCl,0.2g的KCl,1.44g的Na 2 HPO 4和0.24g的KH 2 PO 4
    加入900ml dH 2 O
    将pH调节至7.4
    将最终体积调整为1,000 ml
    在121℃高压灭菌20分钟,灭菌处理
  5. 1 M HEPES(pH 7.5)
    将238.3g HEPES在900ml水中混合
    用NaOH调节pH至7.5 调整到1000毫升
    在121℃高压灭菌20分钟,灭菌处理
  6. 5 M NaCl
    将292g NaCl溶于800ml水中
    调整到最终体积为1000毫升
    在121℃高压灭菌20分钟,灭菌处理
  7. 1 M MgCl 2
    在700ml水中溶解203g的MgCl 2·6H 2 O·6H 2·6H 2 O< br / 调整到最终体积为1000毫升
    在121℃高压灭菌20分钟,灭菌处理
  8. 0.2 M原钒酸钠 在100ml水中溶解3.68g Na 3 VO 4溶液
    涡旋和等分用于存储
    储存于-20°C
  9. 25 mM GTP
    将0.13g GTP加入10ml水中
    使用0.2μm过滤器过滤,并存放等份
    储存于-20°C
  10. 修改MLB缓冲区
    加入50ml水
    2.5ml 1M HEPES(pH7.5)储液
    3ml 5M NaCl储备液
    加入2ml的1M MgCl 2·h/v 10ml甘油 1ml IGEPAL CA-630
    0.2ml 0.5M EDTA
    加入0.5ml的0.2M Na 3 VO 4子/ 0.1ml 25mM GTP储液(以保持RAB21处于其活化状态)
    储存于-20°C

致谢

RAB21活性测定改变自先前公开的研究(Franco等人,2014; Mai等人,2011; Zhu等人, ,2007),并在(Jean等人,2015年)中进行。这项工作是由FRSQ,AHA和CRS博士后研究支持SJ和NIH RO1 GM078176和从SDCSB NIH P50 GM085764到AAK的支持。

参考文献

  1. Franco,I.,Gulluni,F.,Campa,CC,Costa,C.,Margaria,JP,Ciraolo,E.,Martini,M.,Monteyne,D.,De Luca,E.,Germena,G.,Posor ,M。,Maffucci,T.,Marengo,S.,Haucke,V.,Falasca,M.,Perez-Morga,D.,Boletta,A.,Merlo,GRand Hirsch, PI3K II类α控制空间限制性内体PtdIns3P和Rab11活化以促进原发性纤毛功能。 Dev Cell 28(6):647-658。
  2. Jean,S.,Cox,S.,Nassari,S.and Kiger,A.A。(2015)。 饥饿诱导的MTMR13和RAB21活性调节VAMP8以促进自噬体 - 溶酶体融合。 em> EMBO Rep 16(3):297-311。
  3. Mai,A.,Veltel,S.,Pellinen,T.,Padzik,A.,Coffey,E.,Marjomaki,V。和Ivaska,J。 Rab21和p120RasGAP与整联蛋白的竞争性结合调节受体传导和迁移。 J Cell Biol 194(2):291-306。
  4. Zhu,G.,Chen,J.,Liu,J.,Brunzelle,JS,Huang,B.,Wakeham,N.,Terzyan,S.,Li,X.,Rao,Z.,Li, ,XC(2007)。 APPL1 BAR-PH结构域及其与Rab5的相互作用的特征描述。 em> EMBO J 26(14):3484-3493
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。