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Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
采用荧光标记选择性PI3P结合域(PX)测定细胞PI3P含量   

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Abstract

The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.

Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system.

Keywords: Phosphatidylinositol-3-phosphate(phosphatidylinositol-3-phosphate), VPS34(Vps34), PIK3C3(pik3c3), PX domain(PX域), Fluorescence reporter(荧光记者)

Materials and Reagents

  1. ZebaTM spin desalting column (Thermo Fisher Scientific, catalog number: 89891 )
  2. 22 x 22 m cover glasses, Menzel Glaser (VWR, catalog number: 631-1336 )
  3. Millex-GS syringe filter unit, 0.22 µm (Merck Millipore, catalog number: SLGSV255F )
  4. Slide-A-Lyzer dialysis cassettes, 10K MWCO 12 ml (Thermo Fisher Scientific, catalog number: 66810 )
  5. Microscope slides, 75 x 25 mm (superfrost) (VWR, catalog number: 48311-600 )
  6. BL-21 bacteria (New England Biolabs, catalog number: C2530H )
  7. U2OS cells (ATCC, catalog number: HTB-96 )
  8. SOC medium (Thermo Fisher Scientific, catalog number: 15544034 )
  9. LB broth, Miller (Merck Millipore, catalog number: 71753 )
  10. LB agar, Miller (Merck Millipore, catalog number: 1102835000
  11. Ampicillin
  12. IPTG (Sigma-Aldrich, catalog number: I6758 )
  13. Tris (VWR International, catalog number: 103157P )
  14. Sodium chloride (NaCl) (VWR International, catalog number: 27810.364 )
  15. Triton X-100 (Fisher Scientific, catalog number: BP151-500 )
  16. β-mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
  17. PMSF (Sigma-Aldrich, catalog number: P7626 )
  18. Benzamidine (Sigma-Aldrich, catalog number: 12072 )
  19. Brij-35 (Merck Millipore Corporation, catalog number: 1.01894.1000 )
  20. EGTA (Sigma-Aldrich, catalog number: E4378 )
  21. Glutathione sepharose 4B (GE Healthcare, catalog number: 17-0756 )
  22. InstantBlue Coomassie stain (Expedeon, catalog number: ISB1L )
  23. Alexa Fluor 488 TFP ester (Thermo Fisher Scientific, catalog number: A37570 )
  24. Alexa Fluor 594 NHS ester (Thermo Fisher Scientific, catalog number: A37572 )
  25. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  26. HEPES (ForMedium, catalog number: HEPES10 )
  27. Potassium chloride (KCl) (VWR International, catalog number: 26764.260 )
  28. Magnesium acetate (MgAc) (Sigma-Aldrich, catalog number: M5661 )
  29. Potassium glutamate (Sigma-Aldrich, catalog number: G1501 )
  30. DMEM (Thermo Fisher Scientific, Gibco®, catalog number: 11960-044
  31. PBS pH 7.4 (Sigma-Aldrich, catalog number: P4417 )
  32. Bovine serum albumin fraction V (BSA) (Sigma-Aldrich, catalog number: 10735108001 )
  33. Liquid nitrogen
  34. Sodium bicarbonate (Sigma-Aldrich, catalog number: S5761 )
  35. DMSO (Sigma-Aldrich, catalog number: D8418 )
  36. Glycerol, ultra pure (VWR, catalog number: IC800688 )
  37. Sucrose (Sigma-Aldrich, catalog number: S8501 )
  38. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  39. ProLong gold antifade mountant with DAPI (Thermo Fisher Scientific, catalog number: P36931 )
  40. Lysis buffer (see Recipes)
  41. Equilibration buffer (see Recipes)
  42. Wash buffer (see Recipes)
  43. Elution buffer (see Recipes)
  44. Dialysis buffer (see Recipes)
  45. Formaldehyde (see Recipes)
  46. Glutamate buffer (see Recipes)
  47. DMEM + HEPES (see Recipes)
  48. PBS + BSA (see Recipes)

Equipment

  1. Water bath
  2. Bacterial Shaker (e.g., Infors HT)
  3. Benchtop Centrifuge (e.g., Beckman Coulter, model: Allegra X-12R )
  4. Centrifuge (e.g., Beckman Coulter, model: Avanti J-26 XP )
  5. Sonicator (e.g., Sonics Vibra Cell)
  6. Tweezer
  7. GSH-Sepharose beads
  8. Magnetic stir bar (Fisher Scientific)
  9. Gel electrophoresis equipment (e.g., Dual minislab, model: ATTO AE6500 )
  10. Fluorescence microscope (e.g., Nikon Eclipse Ti-E model equipped with appropriate filter set for Alexa-Fluor dye)

Procedure

  1. Purification of GST-PX probe
    The PX domain utilised is M1-R148 of the p40phox protein and has been previously characterised to selectively detect PI(3)P (Kanai et al., 2001). M1-R148 p40phox was cloned into a bacterial (pGEX) expression vector with N-terminal GST tag and linker region (SDLEVLFQGPLGS).
    1. Transform BL21 E.coli cells with 2 ng of plasmid by heat-shock at 42 °C for 45 sec.
    2. Add 900 μl SOC media and incubate in a water bath at 37 °C for 1 h to allow cell recovery.
    3. Spread 100 μl onto LB plates with appropriate selection marker (Ampicillin in the case of pGEX vectors).
    4. Incubate at 37 °C overnight. Plates may be stored at 4 °C before proceeding to the next step.
    5. Inoculate 25 ml of LB media (containing selection marker) with a single colony and incubate for 16 h at 37 °C and agitation at 180 rpm in an Infors HT bacterial shaker. 
    6. Inoculate 500 ml of LB media (containing selection marker) with 10 ml of overnight culture and grow until OD600 = 0.4-0.6 at 37 °C with agitation at 180 rpm in an Infors HT bacterial shaker. Once OD600 is achieved, reduce temperature to 26 °C and induce expression with 10-400 μM IPTG for 16 h (50 μM IPTG gave best results in our hands).
    7. Pellet cells by centrifugation at 4,000 x g for 30 min at 4 °C. The pellet may be stored at -20 °C if required prior to continuing the process.
    8. Add 35 ml of lysis buffer to bacterial pellet and resuspend.
    9. Sonicate at high power on ice, pulse for 15 sec and rest for 15 sec, repeat 3-5 times.
    10. Spin at 15,000 x g for 30 min at 4 °C to clarify lysate and separate the lysate for pelleted cell debris.
    11. Add 10 ml of Equilibration buffer to 3.5 ml (~50% slurry) of GSH-Sepharose, vortex briefly and spin at 1,000 x g for 2 min. Aspirate off supernatant to waste and repeat once more.
    12. Add the washed GSH-Sepharose beads to the clarified lysate and incubate on a roller at 4 °C for 45 min.
    13. Spin down beads at 1,000 x g for 2 min, remove supernatant and add wash buffer up to 40 ml total, vortex briefly and repeat three times.
    14. Add 5 ml of elution buffer to the beads and incubate on ice for 10 min. Spin at 1,000 x g for 2 min and then recover the supernatant. Add 2.5 ml back to the beads and incubate a further 10 min on ice. Spin once more at 1,000 x g for 2 min and recover the supernatant. 
    15. Combine the elutions and pass through a 0.22 μm filter to remove any residual resin.
    16. Determine protein concentration of eluted protein (e.g., by Bradford Assay) and check the purity by running on a polyacrylamide gel and staining with Coomassie blue.
    17. Dialyse the eluted material at 4 °C for 16 h by loading sample into a Slide-A-Lyzer cassette with 10 kDa molecular weight cut off and floating in dialysis buffer with a rotating magnetic stir bar. Dialysis is important to remove glutathione and is better for long-term storage of the protein at -80 °C. Alternately if planning to label the PX protein immediately then it can be dialysed directly into PBS + DTT as required in step 2a. 
  2. Labelling the GST-PX domain with AlexaFluor-594
    The N-terminal GST tag allows the probe to be stained using a GST recognising antibody and appropriate secondary conjugated to a fluorophore. Alternately the GST-PX domain can be labelled directly with a reactive Alexa Fluor dye to reduce the time to stain permeabilised cells.
    The GST-PX domain was labelled utilising a reactive Alexa Fluor (AF) dye, such as AF-594 (A37572) or AF-488 (A37570). Labelling was carried out as recommended in manufacturer’s instructions, briefly:
    1. 0.5 ml of 1 mg/ml purified GST-PX protein was first dialysed with a 10 kDa molecular weight cut off membrane into PBS pH 7.4 + 1 mM DTT for 3 h at 4 °C, the buffer was then changed and dialysed overnight for 16 h at 4 °C. It is important to dialyse the GST-PX protein efficiently as earlier buffer components may interfere with the subsequent labelling efficiency.
    2. 1 M sodium bicarbonate was prepared fresh in ddH2O and 50 µl added to the GST-PX protein solution.
    3. A 100 µg vial of reactive dye was prepared fresh in 10 µl of DMSO and added immediately to the protein and shaken in a thermomixer at 1,100 rpm for 1 h at room temperature. As the dye is light sensitive, cover with aluminium foil to protect the dye as much as possible during the protocol. 
    4. Unconjugated dye was separated from protein by adding sample to a ZebaTM desalting column and centrifugation according to manufacturer’s instructions. After removing unconjugated dye, the protein solution should still retain a colour change indicating that dye has conjugated to the protein. Determining the degree of labelling is not essential but can be calculated by following manufacturer’s instructions available with the Alexa Fluor dye.
    5. Cleared sample was added to glycerol to create a 50% (v/v) solution for long-term storage. The labelled protein should be separated into small aliquots and stored long-term at -80 °C, a working aliquot can be retained at -20 °C. 
    The Alexa FluorTM-PX probe is now complete and ready for use in cellular staining.
  3. Preparation of formaldehyde fixative
    Formaldehyde was prepared in advance from paraformaldehyde powder. Ensure all work is carried out in a fume hood as heating formaldehyde causes the release of toxic gases. For the preparation of 250 ml:
    1. Weigh out 9.25 g of paraformaldehyde and add to 200 ml ddH2O.
    2. Mix with a magnetic stir bar and heat to 60 °C.
    3. Add NaOH dropwise until the solution turns clear.
    4. Remove from heat and add 50 ml of 1 M HEPES (pH 7.4).
    5. Allow to cool before removing from fume hood and store at 4 °C.
  4. Freeze-thaw permeabilisation
    To maintain cellular PI(3)P levels, a detergent-free method of fixing and permeabilizing cells is needed.
    1. Cells to be stained were seeded onto glass coverslips (22 x 22 mm) in 6-well dishes a minimum of 16 h prior to use to allow cells to attach and settle. U2OS cells attach well to glass and settle very flat for clear imaging. Other cell types may require pre-coating of coverslips with gelatin or other reagents.
    2. After treating cells as required for stimuli being investigated, move the 6-well dish onto ice. 
    3. Aspirate media and wash coverslips 2 x 2 ml with ice-cold PBS.
    4. Aspirate PBS and add 2 ml of ice-cold glutamate buffer.
    5. Pick up a single coverslip with tweezers and dab the edge against some paper towel to remove excess buffer. Submerse the coverslip directly into liquid nitrogen until it stops bubbling (~2-3 sec). Remove and place cell side up on paper towel to allow the coverslip to thaw back to room temperature (approximately 1 min or less). During this time it is possible to repeat the freezing procedure with any other coverslips. 
    6. Once the coverslip has thawed, move back into the 6-well dish on ice. 
      Note: Take care with coverslips, after the freeze-thaw coverslips become more brittle and more prone to cracking. 
    7. Wash coverslips 2 x 2 ml with ice-cold glutamate buffer, cells can now be moved off ice and onto bench at room temperature.
    8. Aspirate glutamate buffer and add 2 ml, 3.7% formaldehyde to coverslips for 10 min. After 10 min, remove and replace 2 ml, 3.7% formaldehyde and incubate a further 20 min. 
    9. Wash coverslips 2 x 2 ml with DMEM/HEPES followed by 10 min incubation in 2 ml DMEM/HEPES to quench formaldehyde.
    10. Wash each coverslip 2 x 2 ml and then incubate for 15 min in 2 ml PBS/1% BSA to block. 
    11. Aspirate off buffer and add 100 μl PX-594 probe to each coverslip at 1:200-1:500 (dilution may vary between cell types) in PBS/1% BSA and cover samples with foil from this point on to protect from light. 
    12. Incubate for 1 h at RT.
    13. Wash coverslips 3 x 2 ml with PBS/1% BSA.
    14. Pipette ~10 μl of mounting solution (ProLong Gold Antifade Mountant with DAPI) directly onto a glass slide in a "T" shape. 
    15. Wash each coverslip briefly by dipping into a beaker containing ddH2O, remove excess water by touching the edge against paper towel. Slowly lower the coverslip (cell side down toward the glass slide). Lower gently so that the coverslip comes in contact with the slide from the top of the "T" and downwards.
    16. Ensure all coverslips are covered by foil to protect from light and leave overnight to dry at RT.
    17. Cells can be analysed using a fluorescent microscope with appropriate filters to excite the Alexa-Fluor dye.

Representative data



Figure 1. Example staining with PX-594 in U2OS cells. U2OS cells were treated with VPS34-IN1 inhibitor as indicated for 1 h prior to fixation and staining as detailed here. Bar = 10 μm

Notes

  1. The selectivity of staining for PI(3)P may also be confirmed by generating an additional fluorescently labelled PX omain probe mutated in the Zinc Finger domain (Such as an R57Q mutation) to prevent lipid binding (Kanai et al., 2001). 
  2. The Alexa Fluor dye is light sensitive, so it is important to protect the labelled PX domain probe from light as much as possible by storing in light-resistant tubes and using aluminium foil to protect samples during and after the staining procedure to minimise loss of signal.

Recipes

  1. Lysis buffer
    50 mM Tris (pH 7.5)
    250 mM NaCl
    1% (v/v) Triton X-100
    0.1% β-mercaptoethanol
    0.2 mM PMSF
    1 mM benzamidine
    Store at 4 °C
    Add β-Me, PMSF and benzamidine fresh before use
  2. Wash buffer
    50 mM Tris (pH 7.5)
    250 mM NaCl
    0.03% Brij-35
    0.1 mM EGTA
    0.1% β-mercaptoethanol
    0.2 mM PMSF
    1 mM benzamidine
    Store at RT
    Add β-Me, PMSF and benzamidine fresh before use
  3. Equilibration buffer
    50 mM Tris (pH 7.5)
    250 mM NaCl
    0.1% β-mercaptoethanol
    0.2 mM PMSF
    1 mM benzamidine
    Store at RT
    Add β-Me, PMSF and benzamidine fresh before use
  4. Elution buffer (Make fresh)
    Wash buffer
    20 mM glutathione
    Adjust pH to 7.5
  5. Dialysis buffer (Make fresh)
    50 mM Tris (pH 7.5)
    150 mM NaCl
    0.1 mM EGTA
    270 mM sucrose
    0.03% Brij-35
    0.07% β-mercaptoethanol
    1 mM benzamidine
    0.1 mM PMSF
  6. Glutamate buffer
    25 mM HEPES (pH 7.4)
    25 mM KCl,
    2.5 mM MgAc
    5 mM EGTA
    150 mM potassium glutamate
    Store at 4 °C 
  7. Formaldehyde
    200 mM HEPES (pH 7.4)
    (see protocol for preparation, step 3)
    3.7% (w/v) formaldehyde
    Store at 4 °C
  8. DMEM + HEPES
    DMEM
    10 mM HEPES (pH 7.4)
    Store at 4 °C
  9. PBS + BSA
    PBS
    1% (w/v) BSA
    0.02% sodium azide
    Store at 4 °C

Acknowledgments

This work was supported by the Medical Research Council (MRC) and the pharmaceutical companies supporting the Division of Signal Transduction Therapy Unit (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceutica and Pfizer).

References

  1. Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E., Cantley, L. C. and Yaffe, M. B. (2001). The PX domains of p47phox and p40phox bind to lipid products of PI(3)K. Nat Cell Biol 3(7): 675-678.

简介

脂质磷脂酰肌醇-3-磷酸[PtdIns3P或PI(3)P]扮演许多膜贩运角色,主要由III类PI3K,VPS34产生。然而,确定细胞PI(3)P的水平可以是复杂的。通过甲醇/氯仿提取细胞脂质可能难以分离和鉴定不同的磷脂种类。或者可以使用质谱法,但这需要大量设置专门的设备和时间来利用。
 使用PI(3)P结合特异性重组蛋白结构域是用于确定细胞PI(3)P水平的快速方法,并且还可以允许亚细胞定位的可视化。 p40phox的PX结构域(本文称为PX)对PI(3)P比其它磷脂种类非常特异(Kanai等人,2001)。然而,在细胞中直接表达PX可能是有问题的,因为它将以显性负性方式作用,以比内源蛋白更大的亲和力结合和螯合PI(3)P,从而干扰细胞途径和PI(3)P水平。因此,使用荧光标记的PX后细胞固定更合适,因为它能突出显示PI(3)富集结构,没有扰乱系统的风险。

关键字:phosphatidylinositol-3-phosphate, Vps34, pik3c3, PX域, 荧光记者

材料和试剂

  1. Zeba TM旋转脱盐柱(Thermo Fisher Scientific,目录号:89891)
  2. 22 x 22米盖玻璃,Menzel Glaser(VWR,目录号:631-1336)
  3. Millex-GS注射器过滤单元,0.22μm(Merck Millipore,目录号:SLGSV255F)
  4. Slide-A-Lyzer透析盒,10K MWCO 12ml(Thermo Fisher Scientific,目录号:66810)
  5. 显微镜载片,75×25mm(超级弗罗斯特)(VWR,目录号:48311-600)
  6. BL-21细菌(New England BioLabs,目录号:C2530H)
  7. U2OS细胞(ATCC,目录号:HTB-96)
  8. SOC培养基(Thermo Fisher Scientific,目录号:15544034)
  9. LB肉汤,Miller(Merck Millipore,目录号:71753)
  10. LB琼脂,Miller(Merck Millipore,目录号:1102835000)
  11. 氨苄青霉素
  12. IPTG(Sigma-Aldrich,目录号:I6758)
  13. Tris(VWR International,目录号:103157P)
  14. 氯化钠(NaCl)(VWR International,目录号:27810.364)
  15. Triton X-100(Fisher Scientific,目录号:BP151-500)
  16. β-巯基乙醇(Sigma-Aldrich,目录号:M6250)
  17. PMSF(Sigma-Aldrich,目录号:P7626)
  18. 苄脒(Sigma-Aldrich,目录号:12072)
  19. Brij-35(Merck Millipore Corporation,目录号:1.01894.1000)
  20. EGTA(Sigma-Aldrich,目录号:E4378)
  21. 谷胱甘肽琼脂糖4B(GE Healthcare,目录号:17-0756)
  22. InstantBlue考马斯染色(Expedeon,目录号:ISB1L)
  23. Alexa Fluor 488 TFP酯(Thermo Fisher Scientific,目录号:A37570)
  24. Alexa Fluor 594 NHS酯(Thermo Fisher Scientific,目录号:A37572)
  25. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  26. HEPES(Formedium,目录号:HEPES10)
  27. 氯化钾(KCl)(VWR International,目录号:26764.260)
  28. 将乙酸镁(MgAc)(Sigma-Aldrich,目录号:M5661)
  29. 谷氨酸钾(Sigma-Aldrich,目录号:G1501)
  30. DMEM(Thermo Fisher Scientific,Gibco ,目录号:11960-044
  31. PBS pH 7.4(Sigma-Aldrich,目录号:P4417)
  32. 牛血清白蛋白级分V(BSA)(Sigma-Aldrich,目录号:10735108001)
  33. 液氮
  34. 碳酸氢钠(Sigma-Aldrich,目录号:S5761)
  35. DMSO(Sigma-Aldrich,目录号:D8418)
  36. 甘油,超纯(VWR,目录号:IC800688)
  37. 蔗糖(Sigma-Aldrich,目录号:S8501)
  38. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  39. 具有DAPI的ProLong金抗衰老填充剂(Thermo Fisher Scientific,目录号:P36931)
  40. 裂解缓冲液(见配方)
  41. 平衡缓冲液(见配方)
  42. 洗涤缓冲液(见配方)
  43. 洗脱缓冲液(见配方)
  44. 透析缓冲液(参见配方)
  45. 甲醛(见配方)
  46. 谷氨酸缓冲液(见配方)
  47. DMEM + HEPES(见配方)
  48. PBS + BSA(参见配方)

设备

  1. 水浴
  2. 细菌摇床(例如Infors HT)
  3. 台式离心机(例如,Beckman Coulter,型号:Allegra X-12R)
  4. 离心机(例如,Beckman Coulter,型号:Avanti J-26 XP)
  5. 超声波发生器(例如,Sonics Vibra Cell)
  6. GSH-Sepharose珠
  7. 磁力搅拌棒(Fisher Scientific)
  8. 凝胶电泳设备(例如,Dual minislab,型号:ATTO AE6500)
  9. 荧光显微镜(例如,配备有用于Alexa-Fluor染料的适当过滤器组的Nikon Eclipse Ti-E型)

程序

  1. 纯化GST-PX探针
    所使用的PX结构域是p40phox蛋白的M1-R148,并且之前已经表征为选择性检测PI(3)P(Kanai等人,2001)。 将M1-R148 p40phox克隆到具有N端GST标签和接头区(SDLEVLFQGPLGS)的细菌(pGEX)表达载体中。
    1. 通过在42℃热休克45秒,用2ng质粒转化BL21大肠杆菌细胞。
    2. 加入900μLSOC培养基,并在37℃的水浴中孵育1小时,以允许细胞恢复。
    3. 将100μl铺在具有适当选择标记(在pGEX载体的情况下为氨苄青霉素)的LB平板上。
    4. 在37℃孵育过夜。 板可以在进行下一步骤之前储存在4℃。
    5. 用单个菌落接种25ml LB培养基(含有选择标记),并在Infors HT细菌振荡器中在37℃下以180rpm搅拌孵育16小时。
    6. 用10ml过夜培养物接种500ml LB培养基(含有选择标记),并在Infors HT细菌振荡器中以180rpm搅拌,在37℃下生长直到OD 600 = 0.4-0.6。一旦达到OD 600,将温度降低至26℃,并用10-400μMIPTG诱导表达16小时(50μMIPTG在我们手中给出最佳结果)。
    7. 通过在4℃下以4000xg离心30分钟来沉淀沉淀细胞。如果需要,可以在继续该方法之前将沉淀储存在-20℃。
    8. 加入35毫升裂解缓冲液到细菌沉淀,并重悬。
    9. 在冰上以高功率超声,脉冲15秒,休息15秒,重复3-5次。
    10. 在4℃下以15,000×g离心30分钟以澄清裂解物并分离用于沉淀的细胞碎片的裂解物。
    11. 将10ml平衡缓冲液加入到3.5ml(〜50%浆液)的GSH-Sepharose中,短暂涡旋并以1,000×g旋转2分钟。吸出上清液废物,并重复一次。
    12. 将洗涤的GSH-Sepharose珠添加到澄清的裂解物中,并在滚筒上在4℃下孵育45分钟。
    13. 在1,000×g下旋转珠子2分钟,除去上清液并加入洗涤缓冲液至总共40ml,短暂涡旋并重复三次。
    14. 向珠子中加入5ml洗脱缓冲液,并在冰上孵育10分钟。在1,000×g下旋转2分钟,然后回收上清液。加2.5毫升回珠,并在冰上再孵育10分钟。再次以1,000 x g 再次旋转2分钟,并回收上清液。
    15. 合并洗脱物并通过0.22μm过滤器以除去任何残留的树脂。
    16. 通过Bradford Assay测定洗脱的蛋白质的蛋白质浓度(例如),并通过在聚丙烯酰胺凝胶上电泳并用考马斯蓝染色来检查纯度。
    17. 通过将样品装入具有10kDa分子量的Slide-A-Lyzer盒中,在4℃下透析洗脱的材料16小时,并且用旋转磁力搅拌棒漂浮在透析缓冲液中。透析对于去除谷胱甘肽是重要的并且更好地在-80℃下长期储存蛋白质。或者,如果计划立即标记PX蛋白,则其可以根据步骤2a中的需要直接透析到PBS + DTT中。
  2. 用AlexaFluor-594标记GST-PX结构域
    N末端GST标签允许使用GST识别抗体和适当的二级缀合到荧光团的探针进行染色。或者,可以用反应性Alexa Fluor染料直接标记GST-PX结构域,以减少染色透化细胞的时间。
    使用反应性Alexa Fluor(AF)染料如AF-594(A37572)或AF-488(A37570)标记GST-PX结构域。按照制造商的说明书中的建议进行标记:
    1. 首先用10kDa分子量截留膜将0.5ml 1mg/ml纯化的GST-PX蛋白在4℃下在PBS pH 7.4 + 1mM DTT中透析3小时,然后更换缓冲液并透析过夜16 h。重要的是有效地透析GST-PX蛋白质,因为较早的缓冲液组分可能干扰随后的标记效率。
    2. 在ddH 2 O中新鲜制备1M碳酸氢钠,并将50μl加入到GST-PX蛋白溶液中。
    3. 在10μlDMSO中新鲜制备100μg小瓶的活性染料,并立即加入蛋白质中,并在热混合器中在室温下以1,100rpm振荡1小时。由于染料对光敏感,在方案期间用铝箔覆盖以尽可能多地保护染料。
    4. 通过将样品加入Zeba TM脱盐柱并根据制造商的说明书进行离心,从蛋白质中分离未缀合的染料。除去未偶联的染料后,蛋白质溶液仍应保持颜色变化,表明染料与蛋白质结合。确定标记程度不是必需的,但可以通过以下制造商的使用Alexa Fluor染料的说明书计算。
    5. 将澄清的样品加入到甘油中以产生用于长期储存的50%(v/v)溶液。标记的蛋白质应当分成小等分试样并且在-80℃长期储存,工作等分试样可以保持在-20℃。
    Alexa Fluor TM -PX探针现已完成,可用于细胞染色。
  3. 甲醛固定剂的制备
    预先由多聚甲醛粉末制备甲醛。确保所有的工作在通风橱中进行,因为加热甲醛导致有毒气体的释放。对于250ml的制备:
    1. 称出9.25g多聚甲醛并加入到200ml ddH 2 O中。
    2. 用磁力搅拌棒混合并加热至60℃。
    3. 滴加NaOH直至溶液变澄清。
    4. 从热中取出并加入50ml的1M HEPES(pH7.4)。
    5. 允许冷却,然后从通风橱中取出并存放在4°C。
  4. 冻融渗透
    为了保持细胞PI(3)P水平,需要固定和透化细胞的无洗涤剂方法。
    1. 将待染色的细胞接种在6孔皿中的玻璃盖玻片(22×22mm)上,最少16小时,然后使用以允许细胞附着和沉降。 U2OS细胞很好地附着到玻璃上,并且非常平整以便清晰成像。其他细胞类型可能需要用明胶或其他试剂预涂覆盖玻片。
    2. 根据所研究的刺激需要处理细胞后,将6孔板移至冰上。
    3. 吸出介质和洗玻璃盖2 x 2毫升冰冷PBS。
    4. 吸出PBS,加入2ml冰冷的谷氨酸缓冲液。
    5. 用镊子拿起一个盖玻片,用一些纸巾擦拭边缘,以去除多余的缓冲液。将盖玻片直接浸入液氮,直到停止鼓泡(约2-3秒)。取出并将细胞面朝上放在纸巾上,让盖玻片解冻回到室温(约1分钟或更短)。在此期间,可以使用任何其他盖玻片重复冻结程序。
    6. 一旦盖玻片解冻,移回6孔冰箱上。
      注意:注意盖玻片,冻融盖片变得更脆弱,更容易开裂。 
    7. 用冰冷的谷氨酸盐缓冲液洗涤盖玻片2×2ml,现在可以将细胞从冰上移走并在室温下在台上。
    8. 吸出谷氨酸缓冲液,加2毫升,3.7%甲醛到盖玻片10分钟。 10分钟后,取出并更换2毫升,3.7%甲醛,再培养20分钟。
    9. 用DMEM/HEPES洗涤盖玻片2×2ml,然后在2ml DMEM/HEPES中温育10分钟以猝灭甲醛。
    10. 洗涤每个盖玻片2×2毫升,然后在2毫升PBS/1%BSA孵育15分钟,以封锁。
    11. 吸出缓冲液,并在PBS/1%BSA中以1:200-1:500(稀释可以不同细胞类型)向每个盖玻片添加100μlPX-594探针,并从此点上覆盖样品以保护样品免受光照。  
    12. 在室温下孵育1小时。
    13. 用PBS/1%BSA洗涤盖玻片3×2ml。
    14. 吸取〜10μl的安装解决方案(ProLong金防褪色装载与DAPI)直接在"T"形的玻璃幻灯片。
    15. 通过浸入含有ddH 2 O 2的烧杯中简单地洗涤每个盖玻片,通过使边缘靠在纸巾上来除去多余的水。 慢慢地降低盖玻片(细胞侧朝向玻璃片)。 轻轻下降,使盖玻片从"T"顶部向下与滑块接触。
    16. 确保所有盖玻片由箔覆盖以保护光,并留在夜间在室温干燥。
    17. 可以使用具有适当过滤器的荧光显微镜分析细胞以激发Alexa-Fluor染料。

代表数据



图1.在U2OS细胞中用PX-594染色的实施例。如所指出的,在固定和染色之前,如图所示,用VPS34-IN1抑制剂处理U2OS细胞1小时。 Bar =10μm

笔记

  1. PI(3)P的染色的选择性也可以通过产生在锌指结构域中突变的另外的荧光标记的PX基础探针(例如R57Q突变)来预防脂质结合来确认(Kanai等人。,2001)。
  2. Alexa Fluor染料对光敏感,因此通过储存在耐光管中并使用铝箔保护标记的PX结构域探针免受光,并且在染色程序期间和之后保护样品以使信号损失最小化是重要的 。

食谱

  1. 裂解缓冲液
    50mM Tris(pH7.5) 250mM NaCl 1%(v/v)Triton X-100 0.1%β-巯基乙醇 0.2 mM PMSF
    1mM苄脒
    存储在4°C
    在使用前加入β-Me,PMSF和苄脒
  2. 洗涤缓冲液
    50mM Tris(pH7.5) 250mM NaCl 0.03%Brij-35
    0.1 mM EGTA
    0.1%β-巯基乙醇 0.2 mM PMSF
    1mM苄脒
    在RT存储
    在使用前加入β-Me,PMSF和苄脒
  3. 平衡缓冲器
    50mM Tris(pH7.5) 250mM NaCl 0.1%β-巯基乙醇 0.2 mM PMSF
    1mM苄脒
    在RT存储
    在使用前加入β-Me,PMSF和苄脒
  4. 洗脱缓冲液(新鲜)
    洗涤缓冲液
    20mM谷胱甘肽 调节pH至7.5
  5. 透析缓冲液(新鲜)
    50mM Tris(pH7.5) 150mM NaCl 0.1 mM EGTA
    270mM蔗糖 0.03%Brij-35
    0.07%β-巯基乙醇 1mM苄脒
    0.1mM PMSF
  6. 谷氨酸缓冲液
    25mM HEPES(pH7.4) 25 mM KCl,
    2.5mM MgAc 5mM EGTA
    150mM谷氨酸钾 储存于4°C
  7. 甲醛
    200mM HEPES(pH7.4) (参见制备步骤,步骤3)
    3.7%(w/v)的甲醛 储存于4°C
  8. DMEM + HEPES
    DMEM
    10mM HEPES(pH7.4) 储存于4°C
  9. PBS + BSA
    PBS
    1%(w/v)BSA
    0.02%叠氮化钠 存储在4°C

致谢

这项工作由医学研究理事会(MRC)和支持信号转导治疗单位(AstraZeneca,Boehringer-Ingelheim,GlaxoSmithKline,Merck KGaA,Janssen Pharmaceutica和Pfizer)的制药公司支持。

参考文献

  1. Kanai,F.,Liu,H.,Field,S.J.,Akbary,H.,Matsuo,T.,Brown,G.E.,Cantley,L.C。和Yaffe,M.B。(2001)。 p47phox和p40phox的PX结构域结合PI的脂质产物 3)K。 Nat Cell Biol 3(7):675-678。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Munson, M. J. and Ganley, I. G. (2016). Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX). Bio-protocol 6(16): e1903. DOI: 10.21769/BioProtoc.1903.
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