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[Bio101] PI (phosphoinositide)-3 Kinase Assay
[Bio101] PI肌醇磷脂3-激酶活性测定   

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Abstract

PI3-kinases regulate a wide range of cellular responses through the production of phosphatidylinositol 3, 4, 5-trisphosphate [PI (3, 4, 5) P (3)] in cellular membranes. This protocol provides a highly efficient and easy-to-handle method for sensitive detection and quantification of PI 3-kinase activity.

Materials and Reagents

  1. Tris
  2. NaCl
  3. EDTA
  4. Triton-X100
  5. Glycerol
  6. NaF
  7. PMSF
  8. Aprotinin
  9. Leupeptin
  10. Sepharose
  11. NP-40
  12. Tris-LiCl
  13. TNE
  14. P32 hot ATP (PerkinElmer, catalog number: NEG502A )
  15. MgCl2
  16. MnCl2
  17. EGTA
  18. Methanol
  19. Chloroform
  20. Acetic acid
  21. Isopropanol
  22. Lysis buffer
  23. Pre-washed sepharose
  24. ATP mix (see Recipes)
  25. Phosphoinositol lipid (Avanti Polar Lipid, catalog number: 190082 ) (see Recipes)

Equipment

  1. Centrifuges
  2. Sonicator
  3. Radiation hood
  4. Silica gel TLC plate

Procedure

  1. Lyse the cell with cantley lysis buffer plus protease inhibitor (aprotinin 100 μg/ml, Leupeptin 1 μg/ml, PMSF 35 mg/ml), spin at 10,000 x g for 15 min.
  2. Take the supernatant after centrifugation. Immunoprecipitate with the antibody against protein of interest at 4 °C on wheel overnight.
  3. Add 60 μl pre-washed sepharose (either protein A or protein G) with cut pipet tips to each sample. Put on wheel at 4 °C for 2-3 h.
  4. Spin the sample, (you might want to save the supernatant for future analysis). Wash the samples extensively with following:
    1. 3x 1 ml wash with PBS-1% NP-40
    2. 3x 1 ml wash with Tris- LiCl
    3. 2x 1 ml wash with TNE
    Make sure the washes will not sit at room temperature (RT) for long. Add 60 μl TNE to each sample.
  5. Return the samples on ice while preparing substrates for the assay:
    For each sample phosphoinositol lipid, ATP mix and 100 mM MgCl2 or MnCl2 is needed.
  6. After preparing all the reagents, add 10 μl of PI lipid and 10 μl of 10 mM MgCl2 to each sample. Now you are ready to perform the assay in the radiation hood.
  7. Add hot ATP in the hood. Start the assay by adding 5 μl ATP mix to the sample 10 sec apart and stop each reaction with 20 μl of 8 M HCl when reach 10 min.
    Note: make sure to mix the reaction well for each addition.
  8. Add 160 μl of 1:1 methanol: chloroform to extract lipid and mix well.
  9. Spin the samples in the hot centrifuge for 5 min, remove the upper (water) phase.
  10. Wash the organic layer twice with 200 μl 1 M HCl.
  11. Spot adequate amount of washed chloroform phase onto a silica gel TLC plate (the sample can be stored at -70 °C if not spotting).
  12. Run the plate in the solvent mix (2 M acetic acid/isopropanol 1:2) tank until the solvent front is about 2-3 cm away from the top.
  13. Take out the plate and dry in the hood.
  14. Wrap the plate and expose on the film at -70 °C overnight.
  15. Quantify the phosphatidylinositol3-phosphate spots.

Recipes

For example, if you have 10 samples prepare the reagents as following: Put two extra in: 10+2= 12 samples.

  1. PI lipid
    2 μl x 12 samples= 24 μl
    Take out 24 μl PI, dry under the Argon. Bring up to 120 μl.
    10 mM Tris and 1 mM EGTA.
    Sonicate 10 min.
  2. ATP Mix
    Need total volume 5 μl x 12 samples= 60 μl ATP Mix
    12 μl hot ATP (10 uCi/μl)
    12 μl Cold 10 mM ATP
    36 μl H2O
  3. MgCl2
    10 μl x 12 samples= 120 μl total

References

  1. Wu, H., Shekar, S. C., Flinn, R. J., El-Sibai, M., Jaiswal, B. S., Sen, K. I., Janakiraman, V., Seshagiri, S., Gerfen, G. J., Girvin, M. E. and Backer, J. M. (2009). Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants. Proc Natl Acad Sci U S A 106(48): 20258-20263.
  2. Yip, S. C., El-Sibai, M., Hill, K. M., Wu, H., Fu, Z., Condeelis, J. S. and Backer, J. M. (2004). Over-expression of the p110beta but not p110alpha isoform of PI 3-kinase inhibits motility in breast cancer cells. Cell Motil Cytoskeleton 59(3): 180-188.

简介

PI3-kinases regulate a wide range of cellular responses through the production of phosphatidylinositol 3, 4, 5-trisphosphate [PI (3, 4, 5) P (3)] in cellular membranes. This protocol provides a highly efficient and easy-to-handle method for sensitive detection and quantification of PI 3-kinase activity.

材料和试剂

  1. Tris
  2. NaCl
  3. EDTA
  4. Triton-X100
  5. 甘油
  6. NaF
  7. PMSF
  8. 抑肽酶
  9. 亮肽素
  10. Sepharose
  11. NP-40
  12. Tris-LiCl
  13. TNE
  14. 热ATP(PerkinElmer,目录号:NEG502A)
  15. MgCl 2
  16. MnCl 2
  17. EGTA
  18. 甲醇
  19. 氯仿
  20. 乙酸
  21. 异丙醇
  22. 裂解缓冲液
  23. 预洗琼脂糖
  24. ATP混合(参见配方)
  25. 磷酸肌醇脂质(Avanti Polar Lipid,目录号:190082)(参见配方)

设备

  1. 离心机
  2. 超声波仪
  3. 辐射罩
  4. 硅胶TLC板

程序

  1. 用cantley裂解缓冲液加蛋白酶抑制剂(抑肽酶100μg/ml,亮抑酶肽1μg/ml,PMSF 35mg/ml)裂解细胞,以10,000×g离心15分钟。
  2. 离心后取上清液。 免疫沉淀与感兴趣蛋白质的抗体在4℃下在轮上过夜
  3. 加入60μl预洗琼脂糖凝胶(蛋白质A或蛋白质G),每个样品用切割吸头。 在4°C下放置车轮2-3小时。
  4. 旋转样品,(您可能想保存上清液以备将来分析)。 用以下方法广泛洗涤样品:
    1. 用PBS-1%NP-40洗涤3x 1ml
    2. 3x 1ml用Tris-LiCl洗涤
    3. 2x 1ml用TNE洗涤
    确保洗涤液不会在室温(RT)下长时间放置。 每个样品加入60μlTNE
  5. 将样品放在冰上,同时制备用于测定的底物:
    对于每种样品磷酸肌醇脂质,需要ATP混合物和100mM MgCl 2或MnCl 2。
  6. 在制备所有试剂后,向每个样品中加入10μlPI脂质和10μl10mM MgCl 2。 现在您可以在辐射罩中进行测定。
  7. 在罩中添加热ATP。 通过加入5微升ATP混合物到样品10秒开始测定,停止每个反应与20微升8 M HCl,达到10分钟。
    注意:确保每次添加混合反应良好。
  8. 加入160μl的1:1甲醇:氯仿提取脂质并混匀。
  9. 旋转样品在热离心机5分钟,删除上(水)相。
  10. 用200μl1M HCl洗涤有机层两次。
  11. 将足够量的洗涤的氯仿相点在硅胶TLC板上(如果没有点样,样品可以储存在-70℃)。
  12. 在溶剂混合物(2M乙酸/异丙醇1:2)槽中运行板,直到溶剂前沿离顶部约2-3cm。
  13. 取出板,在罩子里干燥。
  14. 包装板,在-70℃下在膜上暴露过夜
  15. 量化磷脂酰肌醇3-磷酸盐斑点

食谱

例如,如果你有10个样品,准备试剂如下:两个额外的:10 + 2 = 12个样品。

  1. PI脂质
    2μlx 12个样品= 24μl
    取出24μlPI,在氩气下干燥。 可达120μl。
    10mM Tris和1mM EGTA 超声10分钟。
  2. ATP混合物
    需要总体积5μlx 12样品= 60μlATP Mix
    12μl热ATP(10 uCi /μl)
    12μl冷10mM ATP
    36μlH 2 O x/s
  3. MgCl 2
    10μlx 12个样品=总共120μl

参考文献

  1. Wu,H.,Shekar,SC,Flinn,RJ,El-Sibai,M.,Jaiswal,BS,Sen,KI,Janakiraman,V.,Seshagiri,S.,Gerfen,GJ,Girvin,MEand Backer,JM 2009)。 IA类PI3激酶的调节:C2结构域-iSH2结构域接触抑制p85/p110alpha并且在致癌的 p85 突变体中被破坏。 Proc Natl Acad Sci USA 106(48):20258-20263。
  2. Yip,S.C.,El-Sibai,M.,Hill,K.M.,Wu,H.,Fu,Z.,Condeelis,J.S。和Backer,J.M。(2004)。 p110beta 的过表达,但不是 p110alpha < PI3-激酶的同种型抑制乳腺癌细胞中的运动性。 Cell Motil Cytoskeleton 59(3):180-188。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Pang, H. (2012). PI (phosphoinositide)-3 Kinase Assay. Bio-protocol Bio101: e203. DOI: 10.21769/BioProtoc.203;
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salil sukumaran
Institute for Biochemistry I
Why do we need to add extra ATP in addition to Gamma-P32-ATP?
9/15/2014 4:12:14 AM Reply