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May 2018

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Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae
啤酒酵母中的锰超氧化物歧化酶活性测定   

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Abstract

Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O2-) into molecular oxygen (O2) and hydrogen peroxide (H2O2). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature of the cofactor that is required for proper activity. Most eukaryotes, including yeast, possess CuZnSOD and MnSOD. This protocol aims at assessing the activity of the yeast Saccharomyces cerevisiae MnSOD Sod2p from cellular extracts using nitroblue tetrazolium staining. This method can be used to estimate the cellular bioavailability of Mn2+ as well as to evaluate the redox state of the cell.

Keywords: Superoxide dismutase (超氧化物歧化酶), Yeast (酵母), Manganese (锰), Sod2p (Sod2p), Nitroblue tetrazolium (硝基蓝四唑), Redox state (氧化还原状态)

Background

SODs are defined as metal-containing antioxidant enzymes that reduce harmful free radicals of oxygen formed during normal aerobic metabolism to oxygen and hydrogen peroxide. These enzymes are classified based on the metal required as cofactor for proper enzymatic activity: CuZnSODs, MnSODs, FeSODs, and NiSODs. In the yeast Saccharomyces cerevisiae, there are two SODs: the CuZn-Sod1p and the Mn-Sod2p (Abreu and Cabelli, 2010). This protocol focuses on the determination of the enzymatic activity of the Mn-Sod2p, found in the yeast mitochondrial matrix. In this protocol, activity of Sod2p is visualized through nitroblue tetrazolium staining. According to this method, the excitation of riboflavin by light, catalyzed by tetramethylethylenediamine (TEMED), generates superoxide radicals, which convert the yellow nitroblue tetrazolium into blue formazan. In the regions in which Sod2p is present, the superoxide radicals are rapidly removed and formazan formation is prevented. Sod2p is thereby revealed in clear bands on a blue background (Packer, 2002). The method described here includes inhibition of the CuZn-Sod1p by potassium cyanide and thereby enables to determine specifically for the enzymatic activity of the Mn-Sod2p. Apart from providing a method to quickly determine the enzymatic activity of Sod2p, this protocol can be used to correlate the activity of the mitochondrial Sod2p to the bioavailability of manganese cations required for proper activity, a decreased manganese content in the close vicinity of Sod2p resulting in a lower enzymatic activity (Thines et al., 2018). Besides, due to the implication of both Sod2p and manganese cations in resistance against oxidative stress, this protocol can be used to assess the redox state of yeast cells, a decreased enzymatic activity reflecting a reduced ability of the cell to neutralize free radicals.

Materials and Reagents

  1. 425-600 µm diameter acid-washed glass beads (Sigma-Aldrich, catalog number: G8772 )
  2. Petri dishes (Sigma-Aldrich, catalog number: P5606-400EA )
  3. 50 ml Falcon® tubes (Dutscher, catalog number: 352070 )
  4. Eppendorf tubes (VWR, catalog number: 89000-028 )
  5. Bovine serum albumin standard, 2 mg/ml (Thermo Scientific, catalog number: 23210 )
  6. Protease inhibitor cocktail [4 mg/ml of leupeptin (Roth, catalog number: CN33.2 ), aprotinin (Roth, catalog number: A162.3 ), antipain (Roth, catalog number: 2933.2 ), pepstatin (Roth, catalog number: 2936.3 ), and chymostatin (Sigma-Aldrich, catalog number: EI6)]
  7. Yeast extract KAT (Ohly, catalog number: OHLY® KAT)
  8. Glucose (Merck, catalog number: 1083469029 )
  9. Nitroblue tetrazolium (Sigma-Aldrich, catalog member: N6876 )
  10. 4-15% Mini-PROTEAN® TGXTM Precast Protein Gels, 10-well, 50 µl (Bio-Rad, catalog number: 4561084 )
  11. MilliQ water
  12. Ethylenediaminetetraacetic acid (EDTA) disodium salt dihydrate (Sigma-Aldrich, catalog number: E4884 )
  13. Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  14. NaCl (Sigma-Aldrich, catalog number: S9888 )
  15. Triton X-100 (Sigma-Aldrich, catalog number: X100 )
  16. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 10837091001 )
  17. Bicinchoninic acid (Supelco, catalog number: B9643 )
  18. CuSO4·5H2O (Sigma-Aldrich, catalog number: 209198 )
  19. Trizma base (Sigma-Aldrich, catalog number: 93362 )
  20. HCl (Sigma-Aldrich, catalog number: H1758 )
  21. NaOH (Sigma-Aldrich, catalog number: 795429 )
  22. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  23. Bromophenol blue (Sigma-Aldrich, catalog number: B0126 )
  24. Glycine (Sigma-Aldrich, catalog number: 50046 )
  25. TEMED (Sigma-Aldrich, catalog number: T9281 )
  26. Riboflavin (Sigma-Aldrich, catalog number: 47861 )
  27. KCN (Sigma-Aldrich, catalog number: 60178 )
  28. Na2HPO4 (Sigma-Aldrich, catalog number: S7907 )
  29. NaH2PO4 (Sigma, Aldrich, catalog number: S3139 )
  30. K2HPO4 (Sigma-Aldrich, catalog number: 1551128 )
  31. KH2PO4 (Sigma-Aldrich, catalog number: 1551139 )
  32. Liquid nitrogen
  33. YD plates (see Recipes)
  34. NaPO4 buffer (0.1 M, pH 7.8) (see Recipes)
  35. Tris buffer (1 M, pH 6.8) (see Recipes)
  36. Potassium phosphate buffer (1 M, pH 7.8) (see Recipes)
  37. EDTA (100 mM, pH 8.0) (see Recipes)
  38. EGTA (100 mM, pH 8.0) (see Recipes)
  39. Lysis buffer (see Recipes)
  40. CuSO4·5H2O (4%) (see Recipes)
  41. 2x cc. sample buffer for native gels (see Recipes)
  42. Native gel running buffer (see Recipes)
  43. Staining solution (see Recipes)

Equipment

  1. Centrifuge for Eppendorf tubes (Hettich Zentrifugen, model: MIKRO 20 )
  2. Vortex (VWR, model: 444-0996 )
  3. Autoclave (Systec, model: VX/VE )
  4. Protein gel cassette (Bio-Rad, model: 1645052 )
  5. Gel scanner (Amersham, model: 29083461 )
  6. Incubator shaker for yeast growth in liquid culture (Edmund Bühler GmbH, model: VKS-75 control )

Procedure

  1. S. cerevisiae cellular extracts preparation
    1. Streak the S. cerevisiae strains to be analyzed from corresponding glycerol stocks on YD plates (Recipe 1). Incubate for two days at 28 °C.
    2. Using a sterile toothpick, select individual colonies.
    3. Grow yeast cells at 28 °C under agitation (120 rpm) in 50 ml YD medium to an OD600 of 3 (OD600 = 1 corresponds to a density of 1.25 x 107 cells/ml).
      Note: From this step, keep your samples on ice as much as possible.
    4. Centrifuge the yeast culture in 50 ml Falcon® tubes at 3,500 x g at 4 °C for 5 min.
    5. Discard the supernatant and resuspend the pellet in 2 ml ice-cold water.
    6. Centrifuge at 16,000 x g at 4 °C for 20 s.
    7. Discard the supernatant and resuspend the pellet in 200 µl ice-cold lysis buffer (Recipe 7).
    8. Add 200 µg acid-washed 425-600 µm diameter glass beads to the cell suspension.
    9. Perform cell lysis by vortexing 8 x 30 s at 3,000 rpm, with a 15 s break on ice between each vortexing step.
    10. Centrifuge at 2,400 x g at 4 °C for 20 s.
    11. Transfer the supernatant (cell lysate) in a new Eppendorf tube and store it at -80 °C if not used directly, with prior snap freezing in liquid nitrogen.

  2. Protein concentration quantification
    Note: This protocol includes protein quantification using the bicinchoninic acid assay. If familiar with any other method for protein concentration quantification like Bradford assay, this can be used as well.
    1. Prepare standard solutions of bovine serum albumin (BSA) from a 2 mg/ml stock solution according to the Table 1 for establishment of a calibration curve:

      Table 1. Preparation of the BSA standards


    2. Dilute the cell lysates so that their concentration is covered by the calibration curve. If starting from a 50 ml culture harvested at an OD600 of 3, samples can be diluted 10 times (10 µl of cell lysate in 90 µl MilliQ water).
    3. Mix 49 ml bicinchoninic acid with 1 ml CuSO4·5H2O (4%) (Recipe 8).
    4. Mix 20 µl of each standard/sample with 200 µl of the mix bicinchoninic acid/ CuSO4·5H2O.
    5. Incubate for 30 min at 37 °C.
    6. Read the absorbance at 562 nm.
    7. Determine a calibration curve using the standards and deduce the concentration of the samples to be analyzed using this calibration curve. Figure 1 illustrates a typical linear regression that could be obtained with the BSA standards prepared as described above.


      Figure1. Example of calibration curve obtained for the bicinchoninic acid assay from BSA standards prepared as described here. The equation of the linear regression as well as the corresponding R2 are mentioned on the graph.

  3. Sod2p activity staining
    1. Mix a volume of cell lysate corresponding to 200 µg proteins (about 15 µl if starting from a 50 ml culture harvested at an OD600 of 3) with the same volume of 2x cc. sample buffer for native gels (Recipe 9).
    2. Load the resulting mixture on a Mini-PROTEAN® TGXTM precast protein gel.
    3. Run the gel for 4 h at 100 V at 4 °C in native gel running buffer (Recipe 10).
    4. After gel migration, immerge it in 20 ml of a 1 mg/ml nitroblue tetrazolium solution for 15 min under agitation (40 rpm) and in the dark.
    5. Rince the gel with MilliQ water.
    6. Immerge the gel in 25 ml of the staining solution (Recipe 11) for 15 min under agitation (40 rpm) and in the dark.
    7. Rince the gel with MilliQ water.
    8. Expose to light for 15-30 min and scan the gel. The unstained region of the gel corresponds to Sod2p. Figure 2 illustrates the gel obtained for the wild-type yeast strain and for the strains deleted for the genes coding for the CuZn-Sod1p (sod1Δ) or for the Mn-Sod2p (sod2Δ).


      Figure 2. Activity of Sod2p assessed in-gel for the wild-type, sod1Δ, and sod2Δ yeast strains. The intensity of the white band at the level of the arrow correlates with the level of activity of Sod2p (the whiter this region, the higher the activity of Sod2p).

Notes

The intensity of the signal corresponding to the activity of Sod2p can be correlated to the availability of Mn2+ and to the redox status of the cell. In this perspective, a less intense white band on the blue background, reflecting a decreased activity of Sod2p, might be correlated to (i) a decreased bioavailability of Mn2+ in the close vicinity of Sod2p due to its action as cofactor, or to (ii) a reduced cellular ability to resist to oxidative stress due to the implication of Sod2p and manganese cations in neutralizing free radicals. A more quantitative approach can be carried out by quantifying the signal corresponding to Sod2p using any software that is routinely used to quantify Western blotting signals.

Recipes

  1. YD plates
    2 g (2% w/v) yeast extract KAT
    2 g (2% w/v) glucose
    Adjust to 100 ml with MilliQ water and autoclave
    Pour in Petri dishes
  2. NaPO4 buffer (0.1 M, pH 7.8)
    4.48 ml of 1 M Na2HPO4
    0.52 ml of 1 M NaH2PO4
    Adjust to 50 ml with MilliQ water and verify the pH
  3. Tris buffer (1 M, pH 6.8)
    121.14 g Trizma base
    Dilute in approx. 800 ml MilliQ water
    Adjust pH to 6.8 using HCl
    Adjust to a final volume of 1 L with MilliQ water
  4. Potassium phosphate buffer (1 M, pH 7.8)
    14.894 g K2HPO4
    1.972 g KH2PO4
    Adjust to 100 ml with MilliQ water and verify the pH
  5. EDTA (100 mM, pH 8.0)
    3.7224 g EDTA
    Dilute in approx. 80 ml MilliQ water
    Adjust pH to 8.0 using NaOH
    Adjust to a final volume of 100 ml with MilliQ water
  6. EGTA (100 mM, pH 8.0)
    3.8035 g EGTA
    Dilute in approx. 80 ml MilliQ water
    Adjust pH using NaOH
    Adjust to a final volume of 100 ml with MilliQ water
  7. Lysis buffer
    10 ml (10 mM) NaPO4 buffer pH 7.8 (Recipe 2)
    5 ml (5 mM) of 100 mM EDTA
    5 ml (5 mM) of 100 mM EGTA
    0.292 g (50 mM) NaCl
    100 µl (0.1% v/v) Triton X-100
    50 µl Protease inhibitor cocktail
    1 ml (1 mM) of 100 mM phenylmethylsulfonyl fluoride (PMSF) (0.0174 g PMSF in 1 ml ethanol)
    Adjust to a final volume of 100 ml with MilliQ water
  8. CuSO4·5H2O (4%)
    2 g CuSO4·5H2O
    Dilute in 50 ml MilliQ water
  9. 2x cc. sample buffer for native gels
    1.875 ml (62.5 mM) of 1 M Tris-HCl buffer pH 6.8 (Recipe 3)
    12 ml (40% v/v) glycerol
    0.3 ml (0.01% w/v) of 1% (w/v) bromophenol blue (0.5 g bromophenol blue in 50 ml MilliQ water)
    Adjust to 30 ml with MilliQ water
  10. Native gel running buffer
    30.3 g (250 mM) Trizma base
    144.1 g (1.9 M) glycine
    Adjust to 1 L with MilliQ water
    To be diluted 10 times before use
  11. Staining solution
    5 ml (100 mM) potassium phosphate buffer pH 7.8 (Recipe 4)
    162.7 µl (28 mM) TEMED
    0.0005 g (0.028 mM) riboflavin
    0.0163 g (5 mM) KCN
    Adjust to 50 ml with MilliQ water

Acknowledgments

This protocol was adapted from established published procedures (Luk and Culotta, 2001). The work was supported by grants from the Fonds National de la Recherche Scientifique (FNRS, grant PDR-T.0206.16). L.T. is a research fellow at the ‘Fonds pour le Formation à la Recherche dans l’Industrie et dans l’Agriculture’.

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

  1. Abreu, I. A. and Cabelli, D. E. (2010). Superoxide dismutases-a review of the metal-associated mechanistic variations. Biochim Biophys Acta 1804(2): 263-274.
  2. Luk, E. E. and Culotta, V. C. (2001). Manganese superoxide dismutase in Saccharomyces cerevisiae acquires its metal co-factor through a pathway involving the Nramp metal transporter, Smf2p. J Biol Chem 276(50): 47556-47562.
  3. Packer, L. (2002). Superoxide dismutase. United States: Elsevier Science, 197.
  4. Thines, L., Deschamps, A., Sengottaiyan, P., Savel, O., Stribny, J. and Morsomme, P. (2018). The yeast protein Gdt1p transports Mn2+ ions and thereby regulates manganese homeostasis in the Golgi. J Biol Chem, 293(21): 8048-8055.

简介

[摘要 ] 超氧化物歧化酶(SOD能)充当主防御针对反应性氧物质(ROS)通过转换的超氧阴离子自由基(O 2 - )为分子氧(O 2 )和过氧化氢(H 2 ? 2 )。这种酶的家庭成员包括CuZnSODs ,MnSODs ,FeSODs 和NiSODs ,这取决于是需要适当的活动辅助因子的性质。大多数真核生物,包括酵母,都具有CuZnSOD 和MnSOD 。该协议旨在评估酵母的活性 使用硝基蓝四唑染色法从细胞提取物中提取酿酒酵母MnSOD Sod2p 。该方法可用于估计Mn 2+ 的细胞生物利用度以及评估细胞的氧化还原状态。

[背景 ] 的SODs被定义为减少正常有氧代谢为氧气和过氧化氢期间形成的氧的有害自由基含金属的抗氧化剂酶。:这些酶是基于需要作为辅因子进行适当的酶活性的金属分类CuZnSODs ,MnSODs ,FeSODs ,和NiSODs 。在酿酒酵母中,有两个S OD :CuZn-Sod1p和Mn-Sod2p(Abreu和Cabelli ,2010)。该协议的重点是确定在酵母线粒体基质中发现的Mn-Sod2p的酶活性。在此协议中,Sod2p的活性通过硝基蓝四唑鎓染色可视化。根据该方法,在四甲基乙二胺(TEMED)的催化下,光激发核黄素会产生超氧化物自由基,该自由基将黄色的硝基蓝四唑鎓转化为蓝色的甲maz。在其中存在Sod2p的区域中,过氧化物自由基会被快速清除,从而防止了甲maz的形成。因此,Sod2p在蓝色背景上以清晰的条带显示(Packer,2002年)。此处描述的方法包括用氰化钾抑制CuZn-Sod1p,从而能够专门确定Mn-Sod2p的酶活性。除了提供一种快速确定Sod2p酶促活性的方法外,该协议还可用于将线粒体Sod2p的活性与适当活性所需的锰阳离子的生物利用度相关联,从而降低了Sod2p紧邻区域中锰的含量,从而导致较低的酶活性(Thines 等人,2018)。此外,由于Sod2p和锰阳离子均对氧化应激具有抗性,因此该方案可用于评估酵母细胞的氧化还原状态,酶活性降低反映了细胞中和自由基的能力降低。

关键字:超氧化物歧化酶, 酵母, 锰, Sod2p, 硝基蓝四唑, 氧化还原状态

材料和试剂


 


直径425-600 μm的酸洗玻璃珠(Sigma-Aldrich,目录号:G8772)
培养皿(Sigma-Aldrich,目录号:P5606-400EA)
50毫升猎鹰? 管(Dutscher ,目录号:352070)
Eppendorf管S(VWR,目录号:89000-028)
牛血清白蛋白标准品,2 mg / ml(Thermo Scientific,目录号:23210)
蛋白酶抑制剂混合物[ 4 mg / ml亮肽素(Roth,目录号:CN33.2),抑肽酶(Roth,目录号:A162.3),抗痛药(Roth,目录号:2933.2),胃蛋白酶抑制剂(Roth,目录号: 2936.3)和胰凝乳蛋白酶抑制剂(Sigma-Aldrich,目录号:EI6)]
酵母提取物KAT(Ohly ,目录号:OHLY ? KAT)
葡萄糖(默克(Merck),货号:1083469029)
硝基蓝四唑(Sigma-Aldrich,目录成员:N6876)
4- 15%的Mini-PROTEAN ? TGX TM 预制蛋白凝胶,10孔,50微升(Bio-Rad公司,目录号:4561084)
MilliQ 水
乙二胺四乙酸(EDTA)二钠盐d 我水合物(Sigma-Aldrich公司,目录号:E4884)
乙二醇- 双(β- 氨基乙基醚)-N,N,N',N'- 四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
NaCl(Sigma-Aldrich,目录号:S9888)
海卫一X-100(Sigma-Aldrich,目录号:X100)
苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:10837091001)
二辛可宁酸(Supelco公司,目录号:B9643)
CuSO 4 · 5 H 2 O (Sigma-Aldrich,目录号:209198)
Trizma 基地(Sigma-Aldrich,目录号:93362)
HCl(Sigma-Aldrich,目录号:H1758)
NaOH (Sigma-Aldrich,目录号:795429)
甘油(Sigma-Aldrich,目录号:G5516)
溴酚蓝(Sigma-Aldrich,目录号:B0126)
甘氨酸(Sigma-Aldrich,目录号:50046)
TEMED(Sigma-Aldrich,目录号:T9281)
核黄素(Sigma-Aldrich,目录号:47861)
KCN(Sigma-Aldrich,目录号:60178)
Na 2 HPO 4 (Sigma-Aldrich,目录号:S7907)
NaH 2 PO 4 (Sigma,Aldrich,目录号:S3139)
K 2 HPO 4 (Sigma-Aldrich,目录号:1551128)
KH 2 PO 4 (Sigma-Aldrich,目录号:1551139)
液氮
YD板(请参见食谱)
NaPO 4 缓冲液(0.1 M,pH 7.8)(请参见食谱)
Tris缓冲液(1 M,pH 6.8)(请参见配方)
磷酸钾缓冲液(1 M,pH 7.8)(请参见食谱)
EDTA(100 mM,pH 8.0)(请参阅食谱)
EGTA (100 mM,pH 8.0 )(参见食谱)
裂解缓冲液(请参见食谱)
CuSO 4 · 5 H 2 O (4%)(请参阅食谱)
2 x cc。小号上午PLE缓冲区天然凝胶(见食谱)
天然凝胶运行缓冲液(请参见配方)
染色溶液(请参阅食谱)
 


设备


 


Eppendorf管离心机(Hettich Z entrifugen ,型号:MIKRO 20)
涡流(VWR,型号:444-0996)
高压灭菌器(Systec,型号:VX / VE)
蛋白质凝胶盒(Bio-Rad,型号:1645052)
凝胶扫描仪(Amersham ,型号:29083461)
用于液体培养中酵母生长的恒温振荡器(EdmundBühlerGmbH ,型号:VKS-75对照)
 


程序


 


酿酒酵母细胞提取物制备
条纹的酿酒酵母株对从上YD板(配方1)的对应甘油储备进行分析。在28°C下孵育两天。
使用无菌牙签选择单个菌落。
将酵母细胞在50毫升YD培养基中搅拌(120 rpm)在28°C下生长至OD 600 为3(OD 600 = 1对应于1.25 x 10 7 细胞/ ml 的密度)。
注意:从这一步开始,尽量将样品放在冰上。


离心的酵母培养物在50个毫升猎鹰? 在3500管×g下在4 ℃下5分钟。
弃去上清液,将沉淀重悬于2 ml冰冷的水中。
在4°C下以16,000 xg离心20 s。
弃去上清液,将沉淀重悬于200 μl冰冷的裂解缓冲液中(配方7)。
向细胞悬液中加入200 μg酸洗过的425-600 μm直径的玻璃珠。
通过以3,000 rpm 涡旋8 x 30 s 进行细胞裂解,在每个涡旋步骤之间在冰上休息15 s 。
在4°C下以2,400 xg离心20 s。
将上清液(细胞裂解液)转移到新的Eppendorf管中,如果不直接使用,则将其保存在-80 °C,事先在液氮中速冻。
 


蛋白质浓度定量
注意:该方案包括使用双辛可宁酸测定的蛋白质定量。如果熟悉其他任何蛋白质浓度定量方法(例如Bradford分析法),也可以使用此方法。


根据表1 从2 mg / ml储备溶液中制备牛血清白蛋白(BSA)的标准溶液以建立校准曲线:
 


表1 。制定BSA标准





牛血清白蛋白浓度(mg / ml)


H 2 O体积(微升)


BSA体积(微升)


一种


2.000


0


储备溶液300 mg 2 mg / ml





1.500


125


375从储备溶液中2 mg / ml


C


1.000


325


325从储备溶液中2 mg / ml


d


0.750


175


从管B 175


?


0.500


325


来自管C的325


F


0.250


325


E管中的325


G


0.125


325


F管中的325


H


0.025


400


G管100


一世


0


400


0


 


稀释细胞裂解液,使其浓度被校准曲线覆盖。如果来自50ml培养在OD收获开始600 的如图3所示,可将样品稀释10倍(10微升细胞溶胞产物中的90微升中号illiQ 水)。
将49 ml二辛可宁酸与1 ml CuSO 4 · 5 H 2 O (4%)混合(配方8)。
将20 μl的每个标准品/样品与200 μl的二辛可宁酸/ CuSO 4 · 5 H 2 O混合。
在37 °C下孵育30分钟。
读取562 nm处的吸光度。
使用标准品确定校准曲线,并使用该校准曲线推导待分析样品的浓度。图1说明了使用如上所述制备的BSA标准ds 可以获得的典型线性回归。
 






图1。根据本文所述制备的BSA标准品用于二辛可宁酸测定的校准曲线示例。图中提到了线性回归方程以及相应的R 2 。


 


Sod2p活性染色
将相同体积的2 x cc 混合对应于200 μg蛋白质的细胞裂解液(如果从OD 600 为3 收获的50 ml培养物开始,则约为15 μl )。天然凝胶的样品缓冲液(配方9)。   
加载上一个Mini-PROTEAN所得混合物? TGX TM 预制蛋白凝胶。
在天然凝胶电泳缓冲液(配方10)中,在100 V和4°C下将凝胶电泳4 h。
凝胶迁移后,将其在搅拌(40 rpm)和黑暗中浸入20 ml 1 mg / ml 硝基蓝四唑鎓溶液中15分钟。
Rince 与凝胶的MilliQ 水。
在搅拌(40 rpm)和黑暗中,将凝胶浸入25 ml染色溶液(配方11)中15分钟。
Rince 与凝胶的MilliQ 水。
暴露于光下15-30分钟和扫描的凝胶。凝胶的未染色区域对应于Sod2p。图2示出了针对野生型酵母菌株和针对编码CuZn-Sod1p(sod1Δ )或Mn-Sod2p(sod2Δ )的基因而缺失的菌株获得的凝胶。
 






图2.在凝胶中评估的Sod2p对野生型,sod1Δ 和sod2Δ 酵母菌株的活性。箭头处的白带强度与Sod2p的活性水平相关(此区域越白,Sod2p的活性越高)。


 


笔记


 


对应于Sod2p活性的信号强度可以与Mn 2+ 的可用性以及细胞的氧化还原状态相关。从这个角度来看,蓝色背景上强度较弱的白带反映了Sod2p的活性降低,可能与(i )由于其作为辅因子的作用而在Sod2p的附近产生的Mn 2+ 生物利用度降低有关,或者(ii)由于在中和自由基中涉及Sod2p和锰阳离子,导致细胞抵抗氧化应激的能力降低。可以使用任何常规用于定量Western blotting信号的软件对与Sod2p对应的信号进行定量,从而实现更定量的方法。


 


菜谱


 


YD板
2克(2%w / v)酵母提取物KAT


2克(2%w / v)葡萄糖


用M illiQ 水和高压灭菌器调节至100毫升


倒入培养皿


磷酸钠4 b uffer (0.1M,pH7.8)中
4.48毫升1 M Na 2 HPO 4


0.52毫升的1 M NaH 2 PO 4


用M illiQ 水调节至50毫升,并验证pH值


Tris缓冲液(1 M,pH 6.8)
121.14克Trizma 基


约稀释。800毫升M illiQ 水


使用HCl将pH调整至6.8


调整到终体积1升用中号illiQ 水


磷酸钾缓冲液(1 M,pH 7.8)
14.894克K 2 HPO 4


1.972克KH 2 PO 4


用M illiQ 水调节至100毫升并验证pH


EDTA (100毫米,pH 8.0)
3.7224克EDTA


约稀释。80毫升M illiQ 水


使用NaOH将pH调节至8.0


用M illiQ 水调节至100 ml的最终体积


EGTA (100毫米,pH 8.0 )
3.8035克EGTA


约稀释。80毫升M illiQ 水


使用NaOH调节pH


用M illiQ 水调节至100 ml的最终体积


裂解缓冲液
10毫升(10毫米)NaPO 4 缓冲液pH 7.8(配方2)


5毫升(5毫米)的100毫米EDTA


5毫升(5毫米)的100毫米EGTA


0.292 g(50 mM)氯化钠


100微升(0.1%v / v)Triton X-100


50 μl蛋白酶抑制剂混合物


1 ml(1 mM)的100 mM 苯甲基磺酰氟(PMSF)(0.0174 g PMSF在1 ml乙醇中)


用M illiQ 水调节至100 ml的最终体积


CuSO 4 · 5 H 2 O (4%)
2克CuSO 4 · 5 H 2 O


在50毫升M illiQ 水中稀释


2 x cc。天然凝胶样品缓冲液
1.875 ml(62.5 mM)的1 M Tris-HCl缓冲液pH 6.8(配方3)


12毫升(40%v / v)甘油


0.3 ml (0. 01%w / v)的1%(w / v)溴酚蓝(在50 ml M illiQ 水中的0.5 g溴酚蓝)


用M illiQ 水调节至30毫升


? ativ ?凝胶电泳缓冲液
30.3 g(250 mM )Trizma 基础


144.1 g(1.9 M)甘氨酸


调整到1升用中号illiQ 水


使用前需稀释10倍


染色液
5 ml(100 mM)磷酸钾缓冲液pH 7.8(配方4)


162.7微升(28毫米)TEMED


0.0005 g(0.028 mM)核黄素


0.0163 g(5毫米)KCN


用M illiQ 水调节至50毫升


 


致谢


 


该协议改编自已建立的已发布程序(Luk 和Culotta ,2001)。这项工作是由来自全国全宗德拉资金支持RECHERCHE 科学研究(FNRS,授予PDR-T.0206.16)。LT是一个研究员在“全宗倒勒形成点菜RECHERCHE 丹斯欧莱雅工业等丹斯欧莱雅农业”。


 


利益争夺


 


作者声明,他们与本文的内容没有利益冲突。


 


参考文献


 


IA的Abreu和DE的Cabelli (2010)。超氧化物歧化酶-与金属有关的机械变化的综述。Biochim Biophys Acta 1804(2):263-274。
Luk ,EE和Culotta ,VC(2001)。酿酒酵母中的锰超氧化物歧化酶通过涉及Nramp金属转运蛋白Smf2p的途径获得其金属辅因子。 生物化学杂志276(50):47556-47562。
Packer,L。(2002)。超氧化物歧化酶。美国:Elsevier科学,197。
Thines ,L.,Deschamps,A.,Sengottaiyan ,P.,Savel,O.,Stribny ,J.和Morsomme ,P.(2018)。酵母蛋白Gdt1p转运Mn 2+ 离子,从而调节高尔基体中的锰稳态。? 生物学化学293(21) :8048-8055。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Thines, L. and Morsomme, P. (2020). Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae. Bio-protocol 10(5): e3542. DOI: 10.21769/BioProtoc.3542.
  2. Thines, L., Deschamps, A., Sengottaiyan, P., Savel, O., Stribny, J. and Morsomme, P. (2018). The yeast protein Gdt1p transports Mn2+ ions and thereby regulates manganese homeostasis in the Golgi. J Biol Chem, 293(21): 8048-8055.
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