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Quantitative Analysis of Cellular Diacylglycerol Content
细胞中甘油二酯含量的量化分析   

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Abstract

Diacylglycerol (DAG) is a bioactive lipid with diverse biological roles. DAG transiently accumulates in a membrane upon receipt of an appropriate stimulus that activates phospholipase C to cleave phospholipids. The resulting hydrolysis product DAG binds to proteins such as protein kinase C to initiate a variety of downstream cellular processes. DAG kinases attenuate such responses by converting DAG to phosphatidic acid.
This protocol describes an assay designed to quantify cellular DAG levels. The assay exploits the enzymatic conversion of DAG (sn-1,2-diacylglycerol) to phosphatidic acid (1,2-diacyl- sn-glycerol-3-phosphate) in conjunction with the incorporation of a radiolabeled phosphate group by DAG kinase (Figure 1). This assay was described in (Strijbis et al., 2013).


Figure 1. The enzymatic conversion of DAG to phosphatidic acid by DAG kinase

Materials and Reagents

  1. Cells (~2 x 106 cells)
  2. Octyl-β-D-glucoside (Sigma-Aldrich, catalog number: O8001 )
  3. Cardiolipin (Sigma-Aldrich, catalog number: C5646 )
  4. Diethylenetriaminepenta acetic acid (DETAPAC) (Sigma-Aldrich, catalog number: D6518 )
  5. Imidazole (Sigma-Aldrich, catalog number: I5513 )
  6. NaCl (Sigma-Aldrich, catalog number: S7653 )
  7. MgCl2 (Sigma-Aldrich, catalog number: M8266 )
  8. EGTA (Boston Bio Products, catalog number: BM-151 )
  9. DTT (Sigma-Aldrich, catalog number: D0632 )
  10. DAG kinase (Sigma-Aldrich, catalog number: D3065 )
  11. γ33-ATP (PerkinElmer, catalog number: NEG302H001MC )
  12. DAG (Avanti Polar Lipids)
  13. Phosphatidic acid (Sigma-Aldrich, catalog number: P9511 )
  14. Chloroform (Thermo Fisher Scientific, catalog number: C298-50 )
  15. Methanol (Thermo Fisher Scientific, catalog number: BP1105 )
  16. Acetic acid (Sigma-Aldrich, catalog number: 695092 )
  17. N2 gas (Middlesex Gases & Technologies)
  18. Acetone (Thermo Fisher Scientific, catalog number: S70090 )

Equipment

  1. Vortex
  2. Centrifuge
  3. Thin layer chromatography (TLC) (Whatman, catalog number: 4860-820 ) equipment (plates, developing tank)
  4. Phospho-scanner (e.g. Fujifilm Corporation, model: BAS-2500 )
  5. Phospho-imaging screens (e.g. Fujifilm Corporation, model: BAS-MS )

Procedure

  1. Prepare the following solutions before starting the assay
    1. Solubilizing buffer: 7.5% (w/v) octyl-β-D-glucoside and 5 mM cardiolipin in 1 mM diethylenetriaminepenta acetic acid (1 mM DETAPAC, pH 7.0; adjust the pH with NaOH).
    2. 1 mM diethylenetriaminepenta acetic acid (1 mM DETAPAC, pH 6.6; adjust the pH with NaOH).
    3. 2x reaction buffer: 100 mM imidazole HCl, pH 6.6, 100 mM NaCl, 25 mM MgCl2, and 2 mM EGTA.
    4. 100 mM DTT in H2O (prepare fresh).

  2. Sample preparation and total lipid extraction
    1. Wash cells (about 2 x 106) with phosphate-buffered saline (PBS) and extract lipids according to Bligh and Dyer (Bligh et al., 1959).
    2. Dry the chloroform/methanol phase under a gentle flow of N2.

  3. DAG kinase assay
    This assay was adapted from Preiss et al. (1986). Steps from 4 onwards need to be performed in lab areas certified for radioactivity work and the proper lab practice should be followed.
    1. Dissolve the dried lipids in 40 µl of solubilizing buffer by vigorously vortexing for 20 sec.
    2. Incubate the dissolved lipid at room temperature for 10 min.
    3. Keep the samples on ice for 5 min and add 100 µl of 2x reaction buffer, 4 µl of 100 mM DTT and 20 µl of E. coli DAG kinase while keeping the samples on ice.
    4. Initiate the reaction by addition of 3 µCi [γ33]-ATP prepared by dilution in 20 µl of 1 mM DETAPAC (pH 6.6).
    5. After vortexing briefly, incubate the reaction at 25 °C for 30 min.
    6. Stop the reaction by keeping the tubes on ice, and re-extract and dry lipids as described above.
    7. Dissolve the dried lipids in ~50 µl of chloroform/methanol (2/1, v/v).
    8. Apply the lipid extracts on a TLC plate along with lipid standards (Phospatidic acid and DAG).
    9. Run the TLC plate for 30 min by placing it in a TLC chamber that contains about 75 ml of acetone.
    10. Dry the TLC plate and further develop the TLC plate in a chloroform/methanol/acetic acid (65/15/5, v/v/v) solution.
    11. Dry the TLC plate using hair dryer and expose it to phospho-imaging screens.
    12. Detect radiolabeled lipids by scanning the phospho-imaging screen using BAS-2500 scanner.

Acknowledgments

This protocol is adapted from Strijbis et al. (2013). Funding for this work was received from the National Institutes of Health (GM040266; GRF and F32 AI729353; VKV), (H.L.P.), Organization for Scientific Research (KS, MDW, FGT) and the Clay Postdoctoral Fellowship (KS).

References

  1. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8): 911-917.
  2. Preiss, J., Loomis, C. R., Bishop, W. R., Stein, R., Niedel, J. E. and Bell, R. M. (1986). Quantitative measurement of sn-1,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J Biol Chem 261(19): 8597-8600.
  3. Strijbis, K., Tafesse, F. G., Fairn, G. D., Witte, M. D., Dougan, S. K., Watson, N., Spooner, E., Esteban, A., Vyas, V. K., Fink, G. R., Grinstein, S. and Ploegh, H. L. (2013). Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages. PLoS Pathog 9(6): e1003446.

简介

二酰基甘油(DAG)是具有不同生物学作用的生物活性脂质。 DAG在接受激活磷脂酶C以切割磷脂的适当刺激时在膜中瞬时累积。 所得水解产物DAG结合蛋白质例如蛋白激酶C以启动各种下游细胞过程。 DAG激酶通过将DAG转化为磷脂酸来减弱这种应答。该方案描述了设计用于定量细胞DAG水平的测定。 该测定利用DAG(1,2-二酰基甘油)到磷脂酸(1,2-二酰基-sn-甘油-3-磷酸)的酶促转化 结合DAG激酶掺入放射性标记的磷酸基团(图1)。 该测定在(Strijbis等人,2013)中描述。


图1.通过DAG激酶将DAG酶促转化为磷脂酸。

材料和试剂

  1. 细胞(〜2×10 6个细胞)
  2. 辛基-β-D-葡萄糖苷(Sigma-Aldrich,目录号:O8001)
  3. 心磷脂(Sigma-Aldrich,目录号:C5646)
  4. 二亚乙基三胺五乙酸(DETAPAC)(Sigma-Aldrich,目录号:D6518)
  5. 咪唑(Sigma-Aldrich,目录号:I5513)
  6. NaCl(Sigma-Aldrich,目录号:S7653)
  7. MgCl 2(Sigma-Aldrich,目录号:M8266)
  8. EGTA(Boston Bio Products,目录号:BM-151)
  9. DTT(Sigma-Aldrich,目录号:D0632)
  10. DAG激酶(Sigma-Aldrich,目录号:D3065)
  11. γsup 33 -ATP(PerkinElmer,目录号:NEG302H001MC)
  12. DAG(Avanti Polar Lipids)
  13. 磷脂酸(Sigma-Aldrich,目录号:P9511)
  14. 氯仿(Thermo Fisher Scientific,目录号:C298-50)
  15. 甲醇(Thermo Fisher Scientific,目录号:BP1105)
  16. 乙酸(Sigma-Aldrich,目录号:695092)
  17. N 2气体(Middlesex Gases& Technologies)
  18. 丙酮(Thermo Fisher Scientific,目录号:S70090)

设备

  1. 涡流
  2. 离心机
  3. 薄层色谱(TLC)(Whatman,目录号:4860-820)设备(板,显影槽)
  4. Phospho扫描仪(例如,Fujifilm Corporation,型号:BAS-2500)
  5. 磷光成像屏幕(例如,Fujifilm Corporation,型号:BAS-MS)

程序

  1. 在开始测定前准备以下溶液
    1. 增溶缓冲液:在1mM二亚乙基三胺五乙酸(1mM DETAPAC,pH 7.0;用NaOH调节pH)中的7.5%(w/v)辛基-β-D-葡萄糖苷和5mM心磷脂。
    2. 1mM二亚乙基三胺五乙酸(1mM DETAPAC,pH 6.6;用NaOH调节pH)。
    3. 2x反应缓冲液:100mM咪唑HCl,pH 6.6,100mM NaCl,25mM MgCl 2和2mM EGTA。
    4. 100mM DTT的H 2 O(新鲜制备)
  2. 样品制备和总脂质提取
    1. 用磷酸盐缓冲盐水(PBS)洗涤细胞(约2×10 6个),并根据Bligh和Dyer(Bligh等人,1959)提取脂质。
    2. 在N 2缓慢流下干燥氯仿/甲醇相。

  3. DAG激酶测定
    该测定法改编自Preiss等人(1986)。 从4开始的步骤需要在放射性工作认证的实验室区域进行,并且应遵循正确的实验室实践。
    1. 通过剧烈涡旋20秒溶解干燥脂质在40μl增溶缓冲液中。
    2. 在室温下孵育溶解的脂质10分钟。
    3. 将样品保持在冰上5分钟,加入100μl的2x反应缓冲液,4μl的100mM DTT和20μl的E。 大肠杆菌DAG激酶,同时将样品保持在冰上。
    4. 通过加入通过稀释在20μl的1mM DETAPAC(pH 6.6)中制备的3μCi[γ-36] -ATP来引发反应。
    5. 在短暂涡旋后,在25℃下孵育反应30分钟。
    6. 通过将管保持在冰上停止反应,如上所述再提取和干燥脂质
    7. 将干燥的脂质溶解在约50μl氯仿/甲醇(2/1,v/v)中
    8. 将脂质提取物与脂质标准品(磷脂酸和DAG)一起应用于TLC板上
    9. 将TLC板放置在含有约75ml丙酮的TLC室中30分钟
    10. 干燥TLC板并进一步在氯仿/甲醇/乙酸(65/15/5,v/v/v)溶液中展开TLC板。
    11. 使用吹风机干燥TLC板并将其暴露于磷光成像屏
    12. 通过使用BAS-2500扫描仪扫描磷光成像屏幕来检测放射性标记的脂质

致谢

该协议改编自Strijbis等人(2013)。 这项工作的资金来自国家卫生研究院(GM040266; GRF和F32 AI729353; VKV),(H.L.P.),科学研究组织(KS,MDW,FGT)和粘土博士后研究金(KS)。

参考文献

  1. Bligh,E.G。和Dyer,W.J。(1959)。 总脂质提取和纯化的快速方法 Ca 37(8):911-917。
  2. Preiss,J.,Loomis,C.R.,Bishop,W.R.,Stein,R.,Niedel,J.E。和Bell,R.M。(1986)。 定量测量血小板,肝细胞和ras和sis中存在的sn-1,2-二酰基甘油转化的正常大鼠肾细胞。 Biol Chem 261(19):8597-8600。
  3. Strijbis,K.,Tafesse,FG,Fairn,GD,Witte,MD,Dougan,SK,Watson,N.,Spooner,E.,Esteban,A.,Vyas,VK,Fink,GR,Grinstein, ,HL(2013)。 Bruton的酪氨酸激酶(BTK)和Vav1有助于白色念珠菌的Dectin1依赖性吞噬作用/em> 9 9(6):e1003446。
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Copyright: © 2014 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. Tafesse, F. G., Strijbis, K. and Ploegh, H. L. (2014). Quantitative Analysis of Cellular Diacylglycerol Content. Bio-protocol 4(15): e1202. DOI: 10.21769/BioProtoc.1202.
  2. Strijbis, K., Tafesse, F. G., Fairn, G. D., Witte, M. D., Dougan, S. K., Watson, N., Spooner, E., Esteban, A., Vyas, V. K., Fink, G. R., Grinstein, S. and Ploegh, H. L. (2013). Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages. PLoS Pathog 9(6): e1003446.
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