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Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate
采用泛素蛋白或酵素作为底物的非放射性体外PINK1激酶实验

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Abstract

This protocol describes the in vitro phosphorylation of ubiquitin and Parkin by the kinase PINK1 using recombinant proteins. Both substrates, ubiquitin and Parkin, are phosphorylated at the conserved serine 65 residue (pS65-ubiquitin and pS65-Parkin). The protocol also includes the use of monomeric and K48- and K63-linked poly-ubiquitin chains as alternative substrates. Although there are commercially available antibodies, we have not tested their performance in this assay since, but used validated antibodies from our laboratory. An alternative antibody-independent method, the use of phos-tag gels to detect pS65-ubiquitin and pS65-Parkin, is described in addition.

Keywords: PINK1(PINK1), Parkin(帕金), Ubiquitin(泛素), Mitophagy(线粒体自噬), Parkinson's disease(帕金森氏病)

Background

In cells, PINK1 is stabilized and activated by mitochondrial membrane depolarization and other forms of stress that lead to mitochondrial damage. Activated PINK1 phosphorylates ubiquitin, which acts as the receptor for the cytosolic E3 ubiquitin ligase Parkin on the mitochondrial surface. Phosphorylation of Parkin by PINK1 is required for full activity of Parkin towards mitochondrial substrates. The presence of active pS65-Parkin amplifies in a feed-forward mechanism the amount of pS65-ubiquitin on mitochondria, which acts as the mitophagy tag. Eventually, damaged mitochondria are being recognized by autophagy adapters and will be degraded by the proteasome and by autophagy (mitophagy). This crucial mitochondrial quality control pathway promotes turnover of mitochondria and prevents accumulation of dysfunctional mitochondria that can lead to cellular degeneration. Loss-of-function mutations in either PINK1 or Parkin are associated with early-onset Parkinson’s disease.
   In cell-free ubiquitination assays, the presence of pS65-Ub activates the E3 ligase activity of Parkin also in the absence of active PINK1. While many groups use their own purified recombinant proteins for in vitro PINK1 kinase assays, this optimized protocol describes the in vitro phosphorylation of ubiquitin and Parkin with commercially available recombinant proteins.

Materials and Reagents

  1. Recombinant proteins
    1. Active PINK1 [MBP-tagged] from Tribolium castaneum (Ubiquigent, catalog number: 66-0043-050 )
    2. Inactive PINK1 D359A [MBP-tagged] from Tribolium castaneum (Ubiquigent, catalog number: 66-0044-050 )
    3. Parkin (untagged) (Ubiquigent, catalog number: 63-0048-025 )
    4. Ubiquitin (untagged) (BOSTON BIOCHEM, catalog number: U-100H )
    5. Ubiquitin N-terminal biotin (BOSTON BIOCHEM, catalog number: UB-560 )
    6. Biotinylated poly-ubiquitin chains (K48-linked) (BOSTON BIOCHEM, catalog number: UCB-230 )
    7. Poly-ubiquitin chains (K63-linked) (Biotinylated) (BOSTON BIOCHEM, catalog number: UCB-330 )

  2. Chemicals for buffers
    1. HEPES (Sigma-Aldrich, catalog number: H4034 )
    2. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
    3. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
    4. EDTA (EMD Millipore, catalog number: EX0539 )
    5. ATP (AppliChem, catalog number: A1348 )
    6. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M0250 )
    7. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 60370 )
    8. Tris (Santa Cruz Biotchnology, catalog number: sc-3715 )
    9. Hydrochloric acid (37%) (EMD Millipore, catalog number: 1003172500 )
    10. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L4509 )
    11. Glycerol (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP2291 )
    12. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
    13. Bromophenol blue sodium salt (Sigma-Aldrich, catalog number: B5525 )
    14. Glycine (Sigma-Aldrich, catalog number: G7126 )
    15. Methanol (Pharmco-Aaper, catalog number: 339000000 )
    16. Dry milk powder
    17. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
    18. Sodium chloride (NaCl) (Thermo Fisher Scientific, Fisher Scientific, catalog number: BP358-10 )
    19. Tween-20 (Sigma-Aldrich, catalog number: P1379 )
    20. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P3911 )
    21. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S5136 )
    22. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
    23. 10x kinase buffer (see Recipes)
    24. 6x SDS loading buffer (see Recipes)
    25. SDS running buffer (see Recipes)
    26. Blotting buffer (see Recipes)
    27. TBST (see Recipes)
    28. Resolving gel buffer (see Recipes)
    29. 14% resolving phos-tag gel (recipe for mini gel) (see Recipes)
    30. 8% resolving phos-tag gel (recipe for mini gel) (see Recipes)
    31. Stacking gel buffer (see Recipes)
    32. Stacking gel (recipe for two mini gels) (see Recipes)
    33. Phos-tag gel washing buffer 1 (see Recipes)
    34. Phos-tag gel washing buffer 2 (see Recipes)
    35. Phosphate buffer saline (PBS) (see Recipes)
    36. Antigen retrieval solution (see Recipes)

  3. SDS-PAGE/Western blot
    1. 8-16% Tris-glycine gels (for Parkin) (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC60485BOX )
    2. 16% Tris-glycine gels (for ubiquitin) (Thermo Fisher Scientific, InvitrogenTM, catalog number: EC64985BOX )
    3. Pre-stained protein ladder (Bio-Rad Laboratories, catalog number: 1610394 )
    4. PVDF membrane (EMD Millipore, catalog number: IPVH00010 )
    5. pS65-Parkin antibody (Abcam, catalog number: ab154995 )
    6. pS65-Ubiquitin antibody (EMD Millipore, catalog number: ABS1513-I or BOSTON BIOCHEM, catalog number: A-110 )
    7. Monoclonal Parkin antibody Prk8 (Cell Signaling Technology, catalog number: 4211 )
    8. Monoclonal Ubiquitin antibody (LifeSensor, catalog number: VU-101 )
    9. PierceTM streptavidin, HRP-linked (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 21130 )
    10. HRP-conjugated anti-rabbit antibodies (Jackson Immunoresearch, catalog number: 111-035-003 )
    11. HRP-conjugated anti-mouse antibodies (Jackson Immunoresearch, catalog number: 115-035-003 )
    12. Western blot detection reagent (EMD Millipore, catalog number: WBKLS0500 )
    13. X-ray films (Genesee Scientific, catalog number: 30-101L )
    14. Acrylamide:Bis-acrylamide 19:1, solution 40% (w/v) (EMD Millipore, catalog number: 1290-OP )
    15. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 96468 )
    16. Ammonium persulfate [(NH4)2S2O8] (Sigma-Aldrich, catalog number: A9164 )
    17. TEMED (Santa Cruz Biotechnology, catalog number: sc-29111 )
    18. Phos-tagTM acrylamide AAL-107 (Wako Pure Chemical Industries, catalog number: 304-93521 )
    19. 25% glutaraldehyde solution (Sigma-Aldrich, catalog number: G6257 )

Equipment

  1. Standard bench-top centrifuge
  2. Heated shaker for microtubes [e.g., Thermomixer (Eppendorf, catalog number: 5384000020 )]
  3. SDS-PAGE gel electrophoresis and blotting boxes (e.g., VWR International, Peqlab, catalog number: 45-1010-C and Bio-Rad Laboratories, catalog number: 1704070 )
  4. Orbital shaker for Western blots
  5. X-ray film processor or camera detection system (e.g., GE Healthcare, catalog number: 28-9558-10 ) to visualize chemiluminescent Western blot signal

Software

  1. ImageJ or ImageStudio Lite

Procedure

  1. Kinase reaction
    1. Prepare 10x kinase buffer.
      Note: 10x kinase buffer should always be prepared freshly. It can be mixed together from higher concentrated stock solutions. We recommend freezing small (one time use) aliquots of ATP and DTT.
    2. Thaw recombinant proteins on ice.
    3. In a clean microtube, mix PINK1 with ubiquitin (monomers or chains) or Parkin: use 0.1-1 µg of PINK1 per µg of ubiquitin or Parkin.
      Notes:
      1. We recommend using 1 µg of substrate in a 20 µl reaction as a starting point. We typically use 20-100 µl as a total reaction volume.
      2. If biotinylated ubiquitin is used, HRP-coupled streptavidin can be used for detection on phos-tag gels. Otherwise an ubiquitin antibody is necessary.
      3. Compared to poly-ubiquitin chains, free monomeric ubiquitin is simpler to analyze on phos-tag gels since there is only a one band (and therefore only one band-shift). While polymeric K48- and K63-linked chains represent physiological substrates of PINK1 (when attached to mitochondrial substrate proteins), phosphorylated and non-phosphorylated bands may at least partially overlap in particular if chains of various lengths are used.
    4. Optional: Prepare negative control sample as above but use inactive PINK D359A instead of the wild-type enzyme.
    5. Add kinase buffer to a final concentration of 1x.
    6. Spin reactions for 10 sec at 1,000 x g at 4 °C.
    7. Incubate the reactions for 60-120 min (or 24-48 h to achieve 100% substrate phosphorylation) in a heated shaker at 37 °C.
    8. Stop the reactions by adding 6x SDS loading buffer to a final concentration of 1x.
    9. Incubate the reactions for 15 min at 56 °C.
      Note: This lower temperature prevents the unwanted spontaneous formation of ubiquitin 'dimers'.

  2. Detection using phospho-specific antibodies
    1. Assemble SDS gel into an electrophoresis chamber.
    2. Fill the chamber with 1x SDS running buffer.
    3. Load samples on the gel. Use 20-500 ng per lane.
      Note: As a starting point we recommend using 50 ng. Depending on the detection method (antibody, dilution etc.) and equipment the amounts may be changed.
    4. Add 5-10 µl of pre-stained protein ladder in one lane as molecular weight marker.
    5. Run gel using standard conditions (for the gels/equipment listed below use constant 125 Volt for 90 min).
    6. Transfer gel onto methanol-activated PVDF membrane in 1x blotting buffer using standard conditions (for the gels/equipment listed below use constant 100 Volt for 50 min).
    7. Block membrane in 5% dry milk powder in TBST for 1 h at room temperature, shaking.
      Note: We routinely use TBST for Western blots. Other wash buffers might work as well, however phosphate-based buffers are generally not recommended for the detection of phosphorylated proteins.
    8. Incubate membrane with phospho-specific ubiquitin or Parkin antibodies diluted in 5% BSA in TBST overnight at 4 °C.
      Note: The phospho-specific antibodies listed below have not been tested for this particular method. The final concentration of antibody will depend on the amount loaded and must be determined empirically.
    9. Wash membrane 4 x 10 min in TBST at RT.
    10. Incubate membrane with secondary antibodies diluted 1:10,000 in 5% dry milk powder in TBST for one hour at RT.
    11. Wash membrane 4 x 10 min in TBST at RT.
    12. Incubate membrane with detection reagent according to manufacturer’s instructions.
    13. Develop Western blot by exposing X-ray films or using a camera detection system.

  3. Detection using phos-tag gels
    Note: This method is independent of the availability/performance of phospho-specific antibodies. Phosphorylated proteins migrate slower in phos-tag gels compared to non-modified proteins, resulting in a band shift. They can be visualized with specific antibodies against the unmodified protein of interest or with HRP-coupled streptavidin if biotinylated recombinant substrates were used for the kinase reaction.
    1. Prepare phos-tag polyacrylamide gels of desired density: 8% for Parkin and 14% for mono ubiquitin, respectively.
    2. Proceed with SDS-PAGE as above.
      Notes:
      1. Run gels in ice or with cold water perfusion of the apparatus [for the equipment listed below use constant 125 Volt for 90 min (8% gel) and 120-140 min (14% gel), respectively].
      2. Pre-stained protein markers generally cause distortion because they contain EDTA or phosphorylated proteins. At least one lane should be left blank between the marker and the samples. Please note that the molecular weight of pre-stained markers will not be accurate on phos-tag gels.
    3. After electrophoresis incubate phos-tag gels in phos-tag washing buffer 1 for 20 min followed by two incubations, each 20 min, in phos-tag washing buffer 2 at RT.
    4. Transfer gel onto methanol-activated PVDF membrane in 1x blotting buffer (for equipment listed below use constant 105 Volt for 60 min).
      Note: Nitrocellulose membrane might be used instead of PVDF.
    5. For some primary antibodies, antigen retrieval is necessary. Ubiquitin antibody VU-1 requires washing of the membrane with ultrapure water prior to 0.5% glutaraldehyde treatment for 20 min, shaking, followed by three brief washes with PBS.
    6. Block membrane for 1 h at room temperature in 5% dry milk powder in TBST for VU-1 and Prk8 antibodies and in 5% BSA in TBST for streptavidin linked to HRP. Proceed with Western blot as above.
      Note: For 50 ng of substrate use a 1:5,000 dilution of VU-1 antibody to detect ubiquitin and 1:150,000 dilution of Prk8 antibody to detect Parkin. In case of biotinylated ubiquitin, use streptavidin-HRP at a dilution of 1:100,000. For other amounts, dilution of antibodies should be adjusted accordingly. For dilutions > 1:50,000 a predilution is recommended (e.g., dilute antibody 1:100 and then 1:1,000 for a 1:100,000 dilution). If sodium azide is added predilutions can be stored at 4 °C. However, sodium azide will inhibit HRP and is therefore not recommended for streptavidin-HRP.

Data analysis

The here described method uses Western blot analysis as read-out. Phosphorylation by PINK1 over time (Figure 1) can be quantified using densitometric analysis of band intensities using open source software (e.g., ImageJ or ImageStudio Lite). Reactions performed with mutant PINK1 D359A serve as a negative control. On phos-tag gels (Figure 1) the ratio of the shifted versus the non-shifted band allows to calculate the ratio of modified to non-modified substrate. Statistical analysis should be performed with the results from at least three independent experiments.


Figure 1. Kinase assay with N-terminally tagged monomeric ubiquitin using wild-type (WT) or kinase-dead (KD) MBP-TcPINK1. Phosphorylated ubiquitin was detected on a 14% Tris-glycine phos-tag gel with HRP-coupled streptavidin (upper panel) and on a standard 16% Tris-glycine gel with a phospho-specific ubiquitin antibody (pS65-Ub#2) (lower panel).

Recipes

  1. 10x kinase buffer
    Note: Prepare freshly.
    200 mM HEPES, pH 7.4
    100 mM DTT
    1 mM EGTA
    1 mM ATP
    100 mM MgCl2
  2. 6x SDS loading buffer
    Note: Store at -20 °C.
    375 mM Tris, pH 6.8
    9% (w/v) SDS
    50% (v/v) glycerol
    9% (v/v) β-mercaptoethanol
    0.03% (w/v) bromophenol blue
  3. SDS running buffer
    Note: Store at RT.
    25 mM Tris
    200 mM glycine
    0.1% (w/v) SDS
  4. Blotting buffer
    Note: Store at 4 °C.
    25 mM Tris
    192 mM glycine
    0.1% (w/v) SDS
    20% (v/v) methanol
  5. TBST
    Note: Store at RT.
    50 mM Tris, pH 7.4
    150 mM NaCl
    0.1% (v/v) Tween-20
  6. Resolving gel buffer
    Note: Store at 4 °C.
    1.5 M Tris-Cl, pH 8.8
    0.4% (v/v) SDS
  7. 14% resolving phos-tag gel (recipe for one mini gel)
    Note: Prepare freshly.
    2.75 ml resolving gel buffer
    3.875 ml AA:Bis 19:1, solution 40% (w/v)
    4.103 ml water
    111 µl 5 mM phos-tag (final concentration: 50 µM)
    111 µl 10 mM ZnCl2 (final concentration: 100 µM)
    110 µl 10% (w/v) ammonium persulfate in water
    11 µl TEMED
  8. 8% resolving phos-tag gel (recipe for one mini gel)
    Note: Prepare freshly.
    2.75 ml resolving gel buffer
    2.214 ml AA:Bis 19:1, solution 40% (w/v)
    5.764 ml MQ water
    111 µl 5 mM phos-tag (final concentration: 50 µM)
    111 µl 10 mM ZnCl2 (final concentration: 100 µM)
    110 µl 10% (w/v) ammonium persulfate in MQ water
    11 µl TEMED
  9. Stacking gel buffer
    Note: Store at 4 °C.
    0.5 M Tris-Cl, pH 6.8
    0.4% (w/v) SDS
  10. Stacking gel (recipe for two mini gels)
    Note: Prepare freshly.
    2.06 ml stacking gel buffer
    930 µl AA:Bis 19:1, solution 40% (w/v)
    5.17 ml MQ water
    83 µl 10% (w/v) ammonium persulfate in MQ water
    8 µl TEMED
  11. Phos-tag gel washing buffer 1
    Note: Prepare freshly or store at RT.
    1x blotting buffer
    0.01% (w/v) SDS
    1 mM EDTA, pH 8.0
  12. Phos-tag gel washing buffer 2
    Note: Prepare freshly or store at RT.
    1x blotting buffer
    0.01% (w/v) SDS
  13. Antigen retrieval solution
    Note: Prepare freshly from 25% (v/v) glutaraldehyde solution or store at RT.
    PBS
    0.5% (v/v) glutaraldehyde
  14. Phosphate buffer saline (PBS)
    Note: Store at RT.
    137.93 mM NaCl
    2.67 mM KCl
    8.06 mM Na2HPO4
    1.47 mM KH2PO4

Acknowledgments

This protocol was adapted from Fiesel et al. (2015) and Fiesel and Springer (2015). FCF is the recipient of a fellowship from the American Parkinson’s Disease Association. WS was partly supported by the NIH/NINDS R01NS085070, the Michael J. Fox Foundation for Parkinson’s Research, the Foundation for Mitochondrial Medicine, the Mayo Clinic Foundation, the Center for Individualized Medicine, the Center for Regenerative Medicine, the Center for Biomedical Discovery, the Marriott Family Foundation, and a Gerstner Family Career Development Award. WS is a member of a Neuroscience-Focused Research Team at Mayo Clinic Florida.

References

  1. Fiesel, F. C. and Springer, W. (2015). Disease relevance of phosphorylated ubiquitin (p-S65-Ub). Autophagy 11(11): 2125-2126.
  2. Fiesel, F. C., Ando, M., Hudec, R., Hill, A. R., Castanedes-Casey, M., Caulfield, T. R., Moussaud-Lamodiere, E. L., Stankowski, J. N., Bauer, P. O., Lorenzo-Betancor, O., Ferrer, I., Arbelo, J. M., Siuda, J., Chen, L., Dawson, V. L., Dawson, T. M., Wszolek, Z. K., Ross, O. A., Dickson, D. W. and Springer, W. (2015). (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation. EMBO Rep 16(9): 1114-1130.

简介

该协议描述了通过激酶PINK1使用重组蛋白的泛素和帕金蛋白的体外磷酸化。两种底物,泛素和帕金蛋白,在保守的丝氨酸65残基(pS65-泛素和pS65-帕金蛋白)上磷酸化。该方案还包括使用单体和K48和K63连接的聚泛素链作为替代底物。虽然有市售的抗体,我们没有测试他们在这个测定中的性能,因为,但使用我们实验室的验证抗体。另外描述了另一种抗体非依赖性方法,使用phos-标记凝胶检测pS65-泛素和pS65-帕金。

[背景] 在细胞中, PINK1是稳定和激活的线粒体膜去极化和其他形式的应力,导致线粒体损伤。活化的PINK1磷酸化泛素,其作为线粒体表面上胞质E3泛素连接酶Parkin的受体。 Parkin对PINK1的磷酸化是Parkin对线粒体底物的完全活性所必需的。活性pS65-Parkin的存在在前馈机制中扩增了作为线粒体标记的线粒体上的pS65-泛素的量。最终,受损的线粒体被自噬噬菌体衔接子识别,并将被蛋白酶体和自噬(mitophagy)降解。这种关键的线粒体质量控制通路促进线粒体的周转,并防止可导致细胞变性的功能障碍线粒体的积累。 PINK1或Parkin中的功能缺失突变与早发性帕金森病相关。
   在无细胞泛素化测定中,pS65-Ub的存在也在不存在活性PINK1的情况下激活Parkin的E3连接酶活性。虽然许多组使用他们自己的纯化的重组蛋白用于体外PINK1激酶测定,这种优化的方案描述了用市售重组蛋白的泛素和帕金蛋白的体外磷酸化。

关键字:PINK1, 帕金, 泛素, 线粒体自噬, 帕金森氏病

材料和试剂

  1. 重组蛋白
    1. 来自 Tribolium castaneum的活动PINK1 [MBP标记] (Ubiquigent,目录号:66-0043-050)
    2. Inactive PINK1 D359A [MBP-tagged] from Tribolium castaneum (Ubiquigent,目录号:66-0044-050)
    3. Parkin(未标记)(Ubiquigent,目录号:63-0048-025)
    4. 泛素(未标记)(BOSTON BIOCHEM,目录号:U-100H)
    5. 泛素N-末端生物素(BOSTON BIOCHEM,目录号:UB-560)
    6. 生物素化多泛素链(K48连锁)(BOSTON BIOCHEM,目录号:UCB-230)
    7. 聚泛素链(K63连接)(生物素化)(BOSTON BIOCHEM,目录号:UCB-330)

  2. 缓冲剂化学品
    1. HEPES(Sigma-Aldrich,目录号:H4034)
    2. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D0632)
    3. 乙二醇 - 双(2-氨基乙醚)-N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
    4. EDTA(EMD Millipore,目录号:EX0539)
    5. ATP(AppliChem,目录号:A1348)
    6. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M0250)
    7. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:60370)
    8. Tris(Santa Cruz Biotchnology,目录号:sc-3715)
    9. 盐酸(37%)(EMD Millipore,目录号:1003172500)
    10. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L4509)
    11. 甘油(Thermo Fisher Scientific,Fisher Scientific,目录号:BP2291)
    12. β-巯基乙醇(Sigma-Aldrich,目录号:M3148)
    13. 溴酚蓝钠盐(Sigma-Aldrich,目录号:B5525)
    14. 甘氨酸(Sigma-Aldrich,目录号:G7126)
    15. 甲醇(Pharmco-Aaper,目录号:339000000)
    16. 奶粉
    17. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A9647)
    18. 氯化钠(NaCl)(Thermo Fisher Scientific,Fisher Scientific,目录号:BP358-10)
    19. Tween-20(Sigma-Aldrich,目录号:P1379)
    20. 氯化钾(KCl)(Sigma-Aldrich,目录号:P3911)
    21. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S5136)
    22. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
    23. 10x激酶缓冲液(参见配方)
    24. 6x SDS上样缓冲液(见配方)
    25. SDS运行缓冲液(参见配方)
    26. 印迹缓冲液(参见配方)
    27. TBST(参见配方)
    28. 解决凝胶缓冲液(参见配方)
    29. 14%分离的磷标记凝胶(微型凝胶配方)(参见配方)
    30. 8%分离的磷标记凝胶(微型凝胶配方)(参见配方)
    31. 堆叠凝胶缓冲液(参见配方)
    32. 堆叠凝胶(两个迷你凝胶的配方)(参见配方)
    33. Phos标记凝胶洗涤缓冲液1(见配方)
    34. Phos标记凝胶洗涤缓冲液2(见配方)
    35. 磷酸盐缓冲盐水(PBS)(见配方)
    36. 抗原检索溶液(见配方)

  3. SDS-PAGE/Western印迹
    1. 8-16%Tris-甘氨酸凝胶(用于Parkin)(Thermo Fisher Scientific,Invitrogen TM,目录号:EC60485BOX)
    2. 16%Tris-甘氨酸凝胶(用于泛素)(Thermo Fisher Scientific,Invitrogen TM,目录号:EC64985BOX)
    3. 预染色蛋白梯(Bio-Rad Laboratories,目录号:1610394)
    4. PVDF膜(EMD Millipore,目录号:IPVH00010)
    5. pS65-Parkin抗体(Abcam,目录号:ab154995)
    6. pS65-泛素抗体(EMD Millipore,目录号:ABS1513-I或BOSTON BIOCHEM,目录号:A-110)
    7. 单克隆Parkin抗体Prk8(Cell Signaling Technology,目录号:4211)
    8. 单克隆泛素抗体(LifeSensor,目录号:VU-101)
    9. HRP-连接的(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:21130)的Pierce TM链霉亲和素
    10. HRP缀合的抗兔抗体(Jackson Immunoresearch,目录号:111-035-003)
    11. HRP缀合的抗小鼠抗体(Jackson Immunoresearch,目录号:115-035-003)
    12. Western印迹检测试剂(EMD Millipore,目录号:WBKLS0500)
    13. X射线胶片(Genesee Scientific,目录号:30-101L)
    14. 丙烯酰胺:双丙烯酰胺19:1,溶液40%(w/v)(EMD Millipore,目录号:1290-OP)
    15. 氯化锌(ZnCl 2)(Sigma-Aldrich,目录号:96468)
    16. 过硫酸铵[(NH 4)2 SS 2 O 8](Sigma-Aldrich,目录号:A9164 )
    17. TEMED(Santa Cruz Biotechnology,目录号:sc-29111)
    18. Phos-tag TM 丙烯酰胺AAL-107(Wako Pure Chemical Industries,目录号:304-93521)
    19. 25%戊二醛溶液(Sigma-Aldrich,目录号:G6257)

设备

  1. 标准台式离心机
  2. 用于微管的加热摇床[例如,Thermomixer(Eppendorf,目录号:5384000020)]
  3. SDS-PAGE凝胶电泳和印迹盒(例如,VWR International,Peqlab,目录号:45-1010-C和Bio-Rad Laboratories,目录号:1704070)
  4. 用于Western印迹的轨道振动器
  5. X射线胶片处理器或照相机检测系统(例如GE Healthcare,目录号:28-9558-10)以显现化学发光的Western印迹信号

软件

  1. ImageJ或ImageStudio Lite

程序

  1. 激酶反应
    1. 准备10x激酶缓冲液。
      注意:10x激酶缓冲液应始终新鲜制备。它可以从更高浓度的储备溶液混合在一起。我们建议冷冻小(一次使用)等分的ATP和DTT。
    2. 在冰上解冻重组蛋白
    3. 在干净的微管中,将PINK1与泛素(单体或链)或Parkin混合:使用0.1-1μgPINK1 /μg泛素或Parkin。
      注意:
      1. 我们建议在20μl反应中使用1μg底物作为起点。我们通常使用20-100μl作为总反应体积。
      2. 如果使用生物素化的泛素,HRP偶联的链霉亲和素可以用于在磷酸标记凝胶上的检测。否则需要泛素抗体。
      3. 与聚泛素链相比,游离单体泛素更容易在磷标记凝胶上分析,因为只有一个带(因此只有一个带移)。虽然聚合的K48和K63连接的链代表PINK1的生理底物(当连接到线粒体底物蛋白上时),但是如果使用不同长度的链,磷酸化和非磷酸化带可以至少部分重叠。
    4. 可选:如上所述制备阴性对照样品,但使用无活性的PINK D359A代替野生型酶
    5. 加入激酶缓冲液至终浓度为1x
    6. 在4℃下在1,000×g下旋转反应10秒
    7. 在37℃的加热振荡器中孵育反应60-120分钟(或24-48小时以实现100%底物磷酸化)。
    8. 通过加入6x SDS上样缓冲液至终浓度为1x来终止反应。
    9. 在56°C孵育反应15分钟。
      注意:这个较低的温度防止了不需要的自发形成泛素"二聚体"
  2. 使用磷酸特异性抗体的检测
    1. 将SDS凝胶装入电泳室。
    2. 用1×SDS运行缓冲液填充室
    3. 将样品装载到凝胶上。每通道使用20-500 ng。
      注意:作为一个起点,我们建议使用50 ng。根据检测方法(抗体,稀释液等)和设备,可能会改变数量。
    4. 在一个泳道中加入5-10μl预染色的蛋白梯度作为分子量标记
    5. 使用标准条件(对于下面列出的凝胶/设备使用恒定125伏90分钟)运行凝胶
    6. 使用标准条件(对于下面列出的凝胶/设备使用恒定100伏50分钟),在1×印迹缓冲液中将凝胶转移到甲醇活化的PVDF膜上。
    7. 将膜在5%干奶粉中在TBST中在室温下摇动1小时 注意:我们常规使用TBST进行Western印迹。其他洗涤缓冲液也可以工作,但磷酸盐缓冲液通常不推荐用于检测磷酸化蛋白质。
    8. 将膜与在5%BSA的TBST中稀释的磷酸特异性遍在蛋白或Parkin抗体在4℃孵育过夜。
      注意:以下列出的磷酸化特异性抗体尚未针对此特定方法进行测试。抗体的最终浓度取决于加载量,必须根据经验确定。
    9. 在室温下在TBST中洗涤膜4×10分钟
    10. 将膜用在TBST中的5%奶粉中1:10,000稀释的二抗在室温下孵育1小时
    11. 在室温下在TBST中洗涤膜4×10分钟
    12. 根据制造商的说明用检测试剂孵育膜。
    13. 通过暴露X射线胶片或使用相机检测系统来开发Western印迹
  3. 使用phos标签凝胶检测
    注意:该方法不依赖于磷酸特异性抗体的可获得性/性能。与未修饰的蛋白相比,磷酸化蛋白在磷酸标记凝胶中迁移更慢,导致带移。如果生物素化的重组底物用于激酶反应,则可以用针对未修饰的目的蛋白的特异性抗体或用HRP偶联的链霉亲和素来显现它们。
    1. 制备所需密度的phos-tag聚丙烯酰胺凝胶:分别为8%的Parkin和14%的单泛素。
    2. 继续如上所述的SDS-PAGE 注意:
      1. 在冰中或用设备的冷水灌注运行凝胶[对于下面列出的设备分别使用恒定125伏特90分钟(8%凝胶)和120-140分钟(14%凝胶)]。
      2. 预染色的蛋白质标记通常导致畸变,因为它们含有EDTA或磷酸化蛋白质。在标记和样品之间至少要有一个通道留空。请注意,预先染色的标记物的分子量在phos-tag凝胶上将不准确。
    3. 电泳后,在phos-tag洗涤缓冲液1中孵育phos-标签凝胶20分钟,然后在室温下在phos-tag洗涤缓冲液2中孵育两次,每次20分钟。
    4. 在1×印迹缓冲液中将凝胶转移到甲醇活化的PVDF膜上(对于下面列出的设备,使用恒定105伏60分钟)。
      注意:可以使用硝化纤维素膜代替PVDF。
    5. 对于一些一抗,抗原修复是必要的。泛素抗体VU-1需要用超纯水洗涤膜,然后在0.5%戊二醛处理20分钟,摇动,然后用PBS短暂洗涤三次。
    6. 在室温下在TBST中对于VU-1和Prk8抗体在5%干奶粉中和在与TBP连接的链霉亲和素的5%BSA中在室温下封闭膜1小时。继续如上所述的Western印迹 注意:对于50ng底物,使用1:5000稀释的VU-1抗体检测泛素和1:150,000稀释的Prk8抗体检测Parkin。在生物素化的泛素的情况下,使用稀释度为1:100,000的链霉亲和素-HRP。对于其他量,应相应地调整抗体的稀释。对于稀释度> 1:50000,推荐预稀释(例如稀释抗体1:100,然后1:1000稀释)。如果加入叠氮化钠,可以在4℃储存预稀释液。然而,叠氮化钠将抑制HRP,因此不推荐用于链霉亲和素-HRP。

数据分析

这里描述的方法使用蛋白质印迹分析作为读出。可以使用开放源软件(例如ImageJ或ImageStudio Lite),使用条带强度的密度计量分析来定量PINK1随时间的磷酸化(图1)。用突变体PINK1 D359A进行的反应用作阴性对照。在磷标记凝胶(图1)上,移动的与非移动的带的比允许计算修饰的和未修饰的底物的比率。应至少进行三次独立实验的结果进行统计分析

图1.使用野生型(WT)或激酶死亡(KD)MBP-TcPINK1的N-末端标记的单体泛素的激酶测定。在14%Tris-甘氨酸磷酸盐上检测到磷酸化泛素(上图)和在具有磷酸特异性泛素抗体(pS65-Ub#2)的标准16%Tris-甘氨酸凝胶上的标记凝胶(下图).

食谱

  1. 10x激酶缓冲液
    注意:请立即准备。
    200mM HEPES,pH7.4 100 mM DTT
    1 mM EGTA
    1 mM ATP
    100mM MgCl 2/v/v
  2. 6x SDS加样缓冲液
    注意:存储在-20°C。
    375mM Tris,pH 6.8
    9%(w/v)SDS
    50%(v/v)甘油 9%(v/v)β-巯基乙醇 0.03%(w/v)溴酚蓝
  3. SDS运行缓冲区
    注意:存储在RT。
    25 mM Tris
    200mM甘氨酸 0.1%(w/v)SDS
  4. 印迹缓冲区
    注意:存储在4°C。
    25 mM Tris 192 mM甘氨酸 0.1%(w/v)SDS
    20%(v/v)甲醇
  5. TBST
    注意:存储在RT。
    50mM Tris,pH7.4 150mM NaCl 0.1%(v/v)Tween-20
  6. 解决凝胶缓冲液
    注意:存储在4°C。
    1.5M Tris-Cl,pH 8.8
    0.4%(v/v)SDS
  7. 14%分离的磷标记凝胶(一种微型凝胶的配方)
    注意:请立即准备。
    2.75ml分离胶缓冲液
    3.875ml AA:Bis 19:1,溶液40%(w/v) 4.103ml水
    111μl5mM phos-tag(终浓度:50μM) 111μl10mM ZnCl 2(终浓度:100μM)
    110μl10%(w/v)过硫酸铵水溶液
    11μlTEMED
  8. 8%分离的磷标记凝胶(一种微型凝胶的配方)
    注意:请立即准备。
    2.75ml分离胶缓冲液
    2.214ml AA:Bis 19:1,溶液40%(w/v) 5.764毫升MQ水
    111μl5mM phos-tag(终浓度:50μM) 111μl10mM ZnCl 2(终浓度:100μM)
    110μl10%(w/v)过硫酸铵的MQ水溶液
    11μlTEMED
  9. 堆叠凝胶缓冲液
    注意:存储在4°C。
    0.5 M Tris-Cl,pH 6.8
    0.4%(w/v)SDS
  10. 堆叠胶(两个迷你凝胶的配方)
    注意:请立即准备。
    2.06ml堆积凝胶缓冲液
    930μlAA:Bis 19:1,溶液40%(w/v) 5.17毫升MQ水
    83μl10%(w/v)过硫酸铵的MQ水溶液
    8μlTEMED
  11. Phos标记凝胶洗涤缓冲液1
    注意:请立即准备或储存。
    1×印迹缓冲液
    0.01%(w/v)SDS
    1mM EDTA,pH8.0
  12. Phos标记凝胶洗涤缓冲液2
    注意:请立即准备或储存。
    1×印迹缓冲液
    0.01%(w/v)SDS
  13. 抗原回收溶液
    注意:从25%(v/v)戊二醛溶液中新鲜制备或室温储存。
    PBS
    0.5%(v/v)戊二醛
  14. 磷酸盐缓冲液(PBS)
    注意:存储在RT。
    137.93mM NaCl 2.67mM KCl
    8.06mM Na 2 HPO 4
    1.47mM KH 2 PO 4 sub/

致谢

该协议改编自Fiesel等人。 (2015)和Fiesel和Springer(2015)。 FCF是美国帕金森病协会研究金的接受者。 WS的部分支持由NIH/NINDS R01NS085070,迈克尔·福克斯帕金森研究基金会,线粒体医学基金会,梅奥诊所基金会,个体化医学中心,再生医学中心,生物医学发现中心,万豪家庭基金会和Gerstner家庭职业发展奖。 WS是佛罗里达州梅奥诊所的神经科学聚焦研究团队的成员。

参考文献

  1. Fiesel,FC和Springer,W.(2015)。  磷酸化泛素(p-S65-Ub)的疾病相关性。自噬 11(11):2125-2126。
  2. Firesel,FC,Ando,M.,Hudec,R.,Hill,AR,Castanedes-Casey,M.,Caulfield,TR,Moussaud-Lamodiere,EL,Stankowski,JN,Bauer,PO,Lorenzo-Betancor, Ferrer,I.,Arbelo,JM,Siuda,J.,Chen,L.,Dawson,VL,Dawson,TM,Wszolek,ZK,Ross,OA,Dickson,DWand Springer,W。(2015) PINK1依赖性泛素磷酸化的生理相关性的影响。在一些实施方案中,PINK1依赖性泛素磷酸化与PTEN1依赖性泛素磷酸化有关。 a> EMBO Rep 16(9):1114-1130。
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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。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Fiesel, F. C., Hudec, R. and Springer, W. (2016). Non-radioactive in vitro PINK1 Kinase Assays Using Ubiquitin or Parkin as Substrate. Bio-protocol 6(19): e1946. DOI: 10.21769/BioProtoc.1946.
  2. Fiesel, F. C., Ando, M., Hudec, R., Hill, A. R., Castanedes-Casey, M., Caulfield, T. R., Moussaud-Lamodiere, E. L., Stankowski, J. N., Bauer, P. O., Lorenzo-Betancor, O., Ferrer, I., Arbelo, J. M., Siuda, J., Chen, L., Dawson, V. L., Dawson, T. M., Wszolek, Z. K., Ross, O. A., Dickson, D. W. and Springer, W. (2015). (Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation. EMBO Rep 16(9): 1114-1130.
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