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PTEN-lipid Binding Assay
PTEN与脂质结合的试验   

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Abstract

The lipid and protein interactions are an integral and important part of many cellular signaling pathways. The understanding of the selective and specific interaction of the given lipid molecule with the target protein is required for studying cellular signaling. In this assay, different lipids are spotted onto a nitrocellulose membrane to which they attach. Then the membrane is incubated with a lipid binding protein possessing an epitope tag. The protein binds to the lipid which is detected by immunoblotting with an antibody recognizing the epitope tag (see Figure 1). PTEN is an important tumor suppressor which functions as both protein and lipid phosphatase. The primary physiological substrate of PTEN is signaling lipid PtdIns (3, 4, 5) P3, by dephosphrylating PtdIns (3, 4, 5) P3 to PtdIns (4, 5) P2 PTEN negatively regulates PI3K signaling and mediates its tumor-suppressor function by inactivating downstream oncogenic AKT-mediated signaling. The PTEN lipid binding assay is conducted to study the specific binding of PTEN to different lipid molecules.

Keywords: Kavela(kavela), Swapnil(SWAPNIL), Maddika(maddika)



Figure 1. Key steps of the PTEN-lipid binding assay

Materials and Reagents

  1. Lyophilized lipids:
    PE (Sigma-Aldrich, catalog number: P0890 )
    PC (Sigma-Aldrich, catalog number: P1652 )
  2. Hybond C-extra nitrocellulose membrane (Amersham Hybond-ECL, catalog number: RPN303D )
  3. Bacterially Purified GST-fusion protein (GST-PTEN)
  4. Anti-GST monoclonal antibody (Santa Cruz, catalog number: SC-138 )
  5. HRP-conjugated anti mouse secondary antibody (Jackson Immuno Research, catalog number: 315035048 )
  6. ECL (Thermo Scientific Prod, catalog number: 34080 )
  7. Methanol
  8. Chloroform
  9. 1x TBST buffer (see Recipes)
  10. Blocking buffer (see Recipes)

Equipment

  1. Shaker
  2. Film developer

Procedure

  1. Reconstitute the lyophilized lipids in a 2:1:0.8 solution of chloroform: methanol: water to make the required stock (all lipids were constituted to make stock of 1 mM).
  2. Dilute the lipids to get the required working concentration (the working concentration used was 1 nM).
  3. Spot 1 nM of the lipid dilution onto the Hybond C-extra nitrocellulose membrane (each spot is separated by ~1 cm).
  4. Allow to dry at room temperature (RT) for 1 h.
  5. Incubate the membrane with gentle rocking in blocking buffer for 1 h at RT.
  6. Incubate the membrane overnight at 4 °C with gentle rocking in the fresh blocking buffer containing 20-100 nM of the GST-fusion protein (or other epitope tagged protein).
  7. Wash the membrane 10 times over 50 min in TBST (use adequate volume of TBST which will cover the membrane ~10 ml).
  8. Incubate the membrane for 1 h at RT with 1:1,000 dilution of the anti-GST monoclonal antibody in blocking buffer.
  9. Wash the membrane 10 times over 50 min in TBST.
  10. Incubate the membrane for 1 h with a 1:10,000 dilution of the HRP-conjugated antimouse secondary antibody in blocking buffer at RT.
  11. Wash the membrane 12 times over 60 min in TBST.
  12. Detect the lipid binding protein bound to the membrane by ECL according to manufacturer’s instructions (see Figure 2).


    Figure 2. A blot representing the effect of PNUTS on the lipid binding property of PTEN. Nitrocellulose membranes spotted with phophatidylserine (PS) or phosphatidylethanolamine (PE) or phosphatidylcholine (PC) or PS: PE: PC mix (1:1:1) in triplicate was incubated with indicated recombinant proteins. Bound PTEN was detected with anti-GST antibody (adapted from Kavela et al., 2013)

Recipes

  1. 1x TBST solution (1 L)
    50 mM Tris-HCl
    150 mM NaCl
    0.1% Tween 20
    Adjust pH 8 make up to 1 L
  2. Blocking buffer
    3% BSA in 1x TBST

Acknowledgments

This protocol is adapted from Kavela et al. (2013).

References

  1. Kavela, S., Shinde, S. R., Ratheesh, R., Viswakalyan, K., Bashyam, M. D., Gowrishankar, S., Vamsy, M., Pattnaik, S., Rao, S., Sastry, R. A., Srinivasulu, M., Chen, J. and Maddika, S. (2013). PNUTS functions as a proto-oncogene by sequestering PTEN. Cancer Res 73(1): 205-214.

简介

脂质和蛋白质相互作用是许多细胞信号传导途径的整体和重要部分。研究细胞信号传导需要理解给定脂质分子与靶蛋白的选择性和特异性相互作用。在该测定中,将不同的脂质点在它们附着的硝酸纤维素膜上。然后将膜与具有表位标签的脂质结合蛋白一起温育。蛋白质与脂质结合,其通过用识别表位标签的抗体的免疫印迹检测(参见图1)。 PTEN是一种重要的肿瘤抑制剂,其作为蛋白质和脂质磷酸酶起作用。 PTEN的主要生理学底物是信号传导脂质PtdIns(3,4,5)P3,通过将PtdIns(3,4,5)P3去磷酸化为PtdIns(4,5)P2PTEN负调节PI3K信号传导并介导其肿瘤抑制功能通过灭活下游致癌AKT介导的信号。进行PTEN脂质结合测定以研究PTEN与不同脂质分子的特异性结合。

关键字:kavela, SWAPNIL, maddika



图1. PTEN-脂质结合测定的关键步骤

材料和试剂

  1. 冻干脂质:
    PE(Sigma-Aldrich,目录号:P0890) PC(Sigma-Aldrich,目录号:P1652)
  2. Hybond C-extra硝酸纤维素膜(Amersham Hybond-ECL,目录号:RPN303D)
  3. 细菌纯化的GST-融合蛋白(GST-PTEN)
  4. 抗GST单克隆抗体(Santa Cruz,目录号:SC-138)
  5. HRP结合的抗小鼠二抗(Jackson Immuno Research,目录号:315035048)
  6. ECL(Thermo Scientific Prod,目录号:34080)
  7. 甲醇
  8. 氯仿
  9. 1x TBST缓冲区(参见配方)
  10. 阻止缓冲区(参见配方)

设备

  1. 振动器
  2. 电影开发商

程序

  1. 在2:1:0.8的氯仿:甲醇:水的溶液中重构冻干脂质以制备所需的储备液(构成所有脂质以制备1mM的储备液)。
  2. 稀释脂质以获得所需的工作浓度(使用的工作浓度为1nM)
  3. 将1nM的脂质稀释液点在Hybond C-extra硝酸纤维素膜上(每个斑点间隔约1cm)。
  4. 在室温(RT)下干燥1小时
  5. 孵育膜与缓冲摇摆在封闭缓冲液中1小时在室温
  6. 在4℃下孵育膜过夜,在含有20-100nM GST-融合蛋白(或其他表位标记的蛋白质)的新鲜封闭缓冲液中轻轻摇动。
  7. 在TBST中用50分钟清洗膜10次(使用足够体积的TBST,其将覆盖膜〜10ml)。
  8. 在室温下孵育膜1小时,在封闭缓冲液中用抗GST单克隆抗体1:1000稀释。
  9. 在TBST中用50分钟清洗膜10次
  10. 孵育膜1小时与1:10,000稀释的HRP共轭抗小鼠二抗在封闭缓冲液室温。
  11. 在TBST中用60分钟清洗膜12次
  12. 根据制造商的说明,通过ECL检测与膜结合的脂质结合蛋白(参见图2)

    图2.表示PNUTS对PTEN的脂质结合性质的影响的印迹。 用磷脂酰丝氨酸(PS)或磷脂酰乙醇胺(PE)或磷脂酰胆碱(PC)或PS点样的硝化纤维素膜: PE:PC混合物(1:1:1)一式三份与指定的重组蛋白一起孵育。 用抗GST抗体(改编自Kavela等人,2013)检测结合的PTEN。

食谱

  1. 1 TBST溶液(1 L)
    50mM Tris-HCl
    150mM NaCl 0.1%Tween 20
    调节pH 8可达1 L
  2. 阻塞缓冲区
    3%BSA在1x TBST中

致谢

该协议改编自Kavela等人(2013)。

参考文献

  1. Kavela,S.,Shinde,SR,Ratheesh,R.,Viswakalyan,K.,Bashyam,MD,Gowrishankar,S.,Vamsy,M.,Pattnaik,S.,Rao,S.,Sastry,RA,Srinivasulu,M 。,Chen,J.and Maddika,S。(2013)。 PNUTS通过螯合PTEN起到原癌基因的作用。 Cancer Res 73(1):205-214。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kavela, S., Shinde, S. R. and Maddika, S. (2013). PTEN-lipid Binding Assay. Bio-protocol 3(16): e869. DOI: 10.21769/BioProtoc.869.
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