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In Gel Detection of Lipase Activity in Crude Plant Extracts (Olea europaea)
植物粗提物(油橄榄)中脂肪酶活性的凝胶检测   

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Abstract

Here, we provide a detailed protocol describing a SDS-PAGE based procedure to assay in gel neutral lipase activity. Total protein extracts are separated by SDS-PAGE and gels are treated with lipase substrate-α-naphthyl palmitate. This long-chain fatty acid ester is hydrolysed by lipases present in the gel. The product resulting from this reaction can be then visualized in the gel as yellow-brownish activity bands. This relatively simple and effective method of lipase assay detection can be used for crude protein extracts from different plant tissues.

Keywords: Lipase(脂肪酶), Olea europaea(油橄榄), In gel(在凝胶)

Materials and Reagents

  1. Homogenized plant tissues
  2. Liquid nitrogen
  3. 30% acrylamide stock (29: 1 acrylamide: bisacrylamide) (Bio-Rad Laboratories, catalog number: 161-0156 )
  4. TEMED (Bio-Rad Laboratories, catalog number: 161-0801 )
  5. Ammonium persulfate (Sigma-Aldrich, catalog number: A3678 )
  6. 2D Quant Kit (GE Healthcare, catalog number: 80-6483-56 )
  7. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  8. Bromphenol blue (Sigma-Aldrich, catalog number: B0126 )
  9. Glycerol
  10. Triton X-100 (Sigma-Aldrich, catalog number: T8532 )
  11. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  12. NaH2PO4 (Sigma-Aldrich, catalog number: 71505 )
  13. N,N-Dimethylformamide (Fluka, catalog number: 72438 )
  14. Fast blue B salt (Sigma-Aldrich, catalog number: D9805 )
  15. α-naphthyl palmitate (Sigma-Aldrich, catalog number: N9875 )
    Note: α-naphthyl palmitate catalog number: N9875 is no longer available in Sigma-Aldrich, but is available in Santa Cruz Biotechnology, catalog number: CAS 15806-43-6.
  16. Extraction buffer (see Recipes)
  17. 12% separating gel (see Recipes)
  18. 4% stacking gel (see Recipes)
  19. 2 x SDS sample buffer (see Recipes)
  20. 1 M sodium phosphate buffer (see Recipes)
  21. Developing solution (see Recipes)

Equipment

  1. Mortar and pestle
  2. Refrigerated centrifuge 5810R (Eppendorf, catalog number: 5810000.017 ) or similar, equipped with a rotor for 2 ml microcentrifuge tube s
  3. Magnetic stirrer
  4. 2 ml microcentrifuge tube
  5. Tubular glass vials
  6. Mini-PROTEAN® Tetra Cell (Bio-Rad Laboratories, catalog number: 165-8000 )

Software

  1. Quantity One software (Bio-Rad Laboratories)

Procedure

  1. Tissue homogenization and protein extraction
    Note: Do not denature the sample at any time. Proteins must remain in their native, folded state.
    1. Homogenize tissue (0.2 g) to a very fine powder in liquid nitrogen using a precooled mortar and pestle.
    2. Transfer the powder to a 2 ml microcentrifuge tube and add 1.5 ml of extraction buffer and mix well using vortex (see Note 3).
      Note: If samples are used immediately or stored properly it is not necessary to use protease inhibitors.
    3. Stir sample in tubular glass vials using a magnetic stirrer for 1 h at 4 °C.
    4. Centrifuge at 13,500 x g for 30 min at 4 °C.
    5. After centrifugation, collect the supernatant.
    6. Quantify extract and make aliquots.
      Note: The protein concentration was measured using the 2D Quant Kit following the manufacturer’s instructions.

  2. SDS-PAGE
    1. Mix protein sample (approx. 25 μg per sample) with an equal volume of 2x SDS sample buffer. Do not heat at any time.
    2. Load protein sample and perform the SDS-PAGE on a 12% separation gel.

  3. In-gel detection of lipase activity
    1. Remove SDS from the polyacrylamide gels by washing them three times for 30 min each in a solution containing 0.05 M phosphate buffer (pH 7.0) and 2.5 % (v/v) Triton X-100.
    2. Incubate the gels for 30 min at 37 °C in a developing solution in the dark.
    3. Wash gels with distilled water three times for 10 min on a shaker.
    4. The gels can be stored in distilled water up to two months at 4 °C without a significant loss of staining intensity.
    5. To quantify the level of lipase activity, the Quantity One software can be used to measure the density of appearing brown bands.

Recipes

  1. Extraction buffer
    0.5 M sodium phosphate buffer (pH 7.0)
  2. 12% separating gel
    Add the following solutions (total volume: 10 ml)
    30% acrylamide/bisacrylamide
    3 ml
    1.5 M Tris-HCl (pH 8.8)
    2.5 ml
    10% SDS
    100 μl
    dH2O
    4.4 ml
    10% ammonium persulfate
    50 μl
    TEMED
    10 μl
  3. 4% stacking gel
    Add the following solutions (total volume: 5 ml)
    30% acrylamide/bisacrylamide
    0.5 ml
    0.5 M Tris-HCl (pH 6.8)
    1.25 ml
    10% SDS
    50 μl
    dH2O
    3.2 ml
    10% ammonium persulfate
    25 μl
    TEMED
    5 μl
  4. 2 x SDS sample buffer
    100 mM Tris-HCl (pH 6.8)
    4% (w/v) SDS
    0.2% (w/v) bromophenol blue
    20% (v/v) glycerol
  5. 1 M sodium phosphate buffer (pH 7.0)
    57.7 ml 1 M Na2HPO4
    42.3 ml 1 M NaH2PO4
  6. Developing solution
    Mix 40 mg of α-naphthyl palmitate, prepared in 16 ml of N, N-Dimethylformamide with 80 mg of Fast blue BB salt in 144 ml of 0.1 M phosphate buffer (pH 7.0)

Acknowledgments

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced project AGL2008-00517) and the Junta de Andalucía (ERDF-cofinanced project P2010-CVI5767).

References

  1. Rejon, J. D., Zienkiewicz, A., Rodriguez-Garcia, M. I. and Castro, A. J. (2012). Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination. Ann Bot 110(5): 1035-1045.
  2. Zienkiewicz, A., Zienkiewicz, K., Rejon, J. D., Alche Jde, D., Castro, A. J. and Rodriguez-Garcia, M. I. (2014). Olive seed protein bodies store degrading enzymes involved in mobilization of oil bodies. J Exp Bot 65(1): 103-115.

简介

在这里,我们提供详细的协议描述基于SDS-PAGE的程序来测定凝胶中性脂肪酶活性。 通过SDS-PAGE分离总蛋白提取物,并用脂肪酶底物-α-棕榈酸萘酯处理凝胶。 这种长链脂肪酸酯被存在于凝胶中的脂肪酶水解。 由该反应产生的产物然后可以在凝胶中显现为黄棕色活性带。 这种相对简单和有效的脂肪酶测定法可用于来自不同植物组织的粗蛋白提取物。

关键字:脂肪酶, 油橄榄, 在凝胶

材料和试剂

  1. 均质植物组织
  2. 液氮
  3. 30%丙烯酰胺原液(29:1丙烯酰胺:双丙烯酰胺)(Bio-Rad Laboratories,目录号:161-0156)
  4. TEMED(Bio-Rad Laboratories,目录号:161-0801)
  5. 过硫酸铵(Sigma-Aldrich,目录号:A3678)
  6. 2D Quant Kit(GE Healthcare,目录号:80-6483-56)
  7. (Sigma-Aldrich,目录号:T1503)
  8. 溴酚蓝(Sigma-Aldrich,目录号:B0126)
  9. 甘油
  10. Triton X-100(Sigma-Aldrich,目录号:T8532)
  11. Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  12. NaH 2 PO 4(Sigma-Aldrich,目录号:71505)
  13. N,N-二甲基甲酰胺(Fluka,目录号:72438)
  14. 快蓝B盐(Sigma-Aldrich,目录号:D9805)
  15. 棕榈酸α-萘酯(Sigma-Aldrich,目录号:N9875) - 棕榈酸萘酯目录号:N9875不再适用于Sigma-Aldrich,但可在Santa Cruz Biotechnology,目录号:CAS 15806- 43-6。
  16. 提取缓冲液(参见配方)
  17. 12%分离凝胶(见配方)
  18. 4%堆积凝胶(见配方)
  19. 2 x SDS样品缓冲液(见配方)
  20. 1 M磷酸钠缓冲液(见配方)
  21. 开发解决方案(参见配方)

设备

  1. 砂浆和杵
  2. 配备有用于2ml微量离心管的转子的冷冻离心机5810R(Eppendorf,目录号:5810000.017)或类似物
  3. 磁力搅拌器
  4. 2 ml微量离心管
  5. 管状玻璃小瓶
  6. Mini-PROTEAN Tetra Cell(Bio-Rad Laboratories,目录号:165-8000)

软件

  1. Quantity One软件(Bio-Rad Laboratories)

程序

  1. 组织匀浆和蛋白质提取
    注意:不要在任何时候使样品变性。 蛋白质必须保持其天然的折叠状态。
    1. 使用预冷的研钵和杵将组织(0.2g)均质化成在液氮中的非常细的粉末。
    2. 转移粉末到2毫升微量离心管,加入1.5毫升   提取缓冲液,并使用涡旋混合均匀(见注3) 注意:如果样品立即使用或正确储存,则不需要使用蛋白酶抑制剂。
    3. 使用磁力搅拌器在4℃下在管状玻璃小瓶中搅拌样品1小时。
    4. 在4℃下以13,500×g离心30分钟。
    5. 离心后,收集上清液。
    6. 量化提取物并制成等分试样。
      注意:使用2D Quant Kit按照制造商的说明测量蛋白质浓度。

  2. SDS-PAGE
    1. 将蛋白质样品(每个样品约25μg)与等体积的2x SDS样品缓冲液混合。 不要随时加热。
    2. 加载蛋白质样品并在12%分离凝胶上进行SDS-PAGE。

  3. 脂肪酶活性的凝胶检测
    1. 通过洗涤从聚丙烯酰胺凝胶中除去SDS三次 在含有0.05M磷酸盐缓冲液(pH7.0)的溶液中各30分钟, 和2.5%(v/v)Triton X-100
    2. 在37℃下在黑暗中的显影溶液中孵育凝胶30分钟。
    3. 用蒸馏水在振荡器上洗涤凝胶三次,每次10分钟。
    4. 凝胶可以在4℃下在蒸馏水中保存两个月,而染色强度没有显着损失
    5. 为了量化脂肪酶活性的水平,使用Quantity One软件 可用于测量出现棕色带的密度。

食谱

  1. 提取缓冲区
    0.5M磷酸钠缓冲液(pH7.0)
  2. 12%分离凝胶
    加入以下溶液(总体积:10ml)
    30%丙烯酰胺/双丙烯酰胺 3 ml
    1.5 M Tris-HCl(pH 8.8)
    2.5 ml
    10%SDS
    100微升
    dH 2 2 O 4.4 ml
    10%过硫酸铵
    50微升
    TEMED
    10微升
  3. 4%堆积胶
    加入以下溶液(总体积:5ml)
    30%丙烯酰胺/双丙烯酰胺 0.5 ml
    0.5M Tris-HCl(pH 6.8)
    1.25 ml
    10%SDS
    50微升
    dH 2 2 O 3.2 ml
    10%过硫酸铵
    25μl
    TEMED
    5微升
  4. 2 x SDS样品缓冲液
    100 mM Tris-HCl(pH 6.8)
    4%(w/v)SDS
    0.2%(w/v)溴酚蓝
    20%(v/v)甘油
  5. 1M磷酸钠缓冲液(pH 7.0) 57.7ml 1M Na 2 HPO 4
    42.3ml 1M NaH 2 PO 4 dub/d
  6. 开发解决方案
    将40mg在16ml N,N-二甲基甲酰胺中制备的棕榈酸α-萘酯与80mg的快蓝BB盐在144ml的0.1M磷酸盐缓冲液(pH7.0)中混合,

致谢

这项工作得到了西班牙科学和创新部(MICINN)(ERDF-共同融资项目AGL2008-00517)和Junta deAndalucía(ERDF-共同融资项目P2010-CVI5767)的支持。

参考文献

  1. Rejon,J.D.,Zienkiewicz,A.,Rodriguez-Garcia,M.I。和Castro,A.J。(2012)。 橄榄油(
  2. Zienkiewicz,A.,Zienkiewicz,K.,Rejon,J.D.,Alche Jde,D.,Castro,A.J。和Rodriguez-Garcia,M.I。(2014)。 橄榄种子蛋白体储存降解酶,参与油体的动员。 J Exp Bot 65(1):103-115。
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引用:Zienkiewicz, A., Rejón, J. D., Zienkiewicz, K., Castro, A. J. and Rodríguez-García, M. I. (2015). In Gel Detection of Lipase Activity in Crude Plant Extracts (Olea europaea). Bio-protocol 5(8): e1444. DOI: 10.21769/BioProtoc.1444.
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