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Phosphoinositides Coated Beads Binding Assay
磷酸肌醇包衣颗粒结合试验   

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Abstract

The PIs coated beads assay or “PIP-Beads” developed by Echelon Biosciences (Salt Lake City, USA) is a quick assay to recognize which PIs are able to bind to a given protein domain, in a quantitative way. It is much faster and cheaper than liposomes and more reproducible than PIP-strip assays. The “PIP-Beads” assay is a biochemical assay that basically involves an incubation of a purified protein or protein domain with the appropriate PI-coated set of beads. After washing, drying and resuspending the samples, they can be easily analyzed by SDS-PAGE separation. Phosphoinositides (PIs) have been characterized as important determinants of cell membrane domains, such as the apical and basolateral domains in epithelial polarized cells (Martin-Belmonte and Mostov, 2007), controlling membrane trafficking (Szentpetery et al., 2010) or determining the presynaptic or postsynaptic terminal in neurons, among other functions (Di Paolo and De Camilli, 2006). These phosphoinositides enriched membranes bring the proteomic machinery together, confers to these membrane their different identities and functions. This protein-PIs interaction in many cases involves direct binding of specific protein membrane domains with certain PIs. Some of these domains are characterized such as PH domains from phospholipase-C- or synaptotagmin-like C2 domains (Galvez-Santisteban et al., 2012), while some of them are not. To determine which PI is binding to a given protein domain, it is important to have a quick and efficient assay. The liposome binding assays are very good to establish the kinetic properties of binding, but they are expensive and permit only to test a few PIs per experiment. On the other hand, PIP-strip (phosphatidil-inositol-phosphate) based analysis is easy and fast, however the PIs are presented in a flat surface and the reproducibility is sometimes limited.

Materials and Reagents

  1. PIP-Beads (Echelon) with the corresponding phosphoinositide of interest. Echelon provides a wide range of PI and other phospholipid coated beads on demand, including a sample pack that has a wide range of PIP-coated beads (P-B00S).
  2. 2 µg of purified GST-tagged protein per tube of beads and GST alone for control
    The protein can be produced and purified by standard methods of GST-tagged protein purification by glutathione-sepharose beads. For the present assay, it is recommended to elute the protein from glutathione-sepharose beads and quantify it before use. Moreover, it is important to use high concentrations of protein, in order to use the smallest possible volume.
  3. HEPES
  4. Lgepal CA630
  5. SDS
  6. Glycerol
  7. Tris-HCl
  8. β-mercaptoethanol
  9. Bromophenol blue
  10. Wash/binding buffer (see Recipes)
  11. 2x Reducing Laemmli Sample Buffer (see Recipes)

Equipment

  1. Refrigerated centrifuge
  2. Oscillatory or gyratory shaker
  3. Aspiration pump
  4. Needles 0.7 x 30 mm
  5. 1.5 ml Tubes

Procedure

  1. Before starting
    a. Prepare or thaw the purified GST-protein.
    b. Pre-cool the centrifuge.
    c. Prepare the wash/binding buffer.
    d. Make aspiration tips by cutting the tip of a needle with pliers or a similar tool (Figure 1). This will minimize the needle diameter.


    Figure 1. Aspiration tips

  2. Use 50 µl of PI-beads per protein. Echelon provide beads suspended in buffer 50/50.
  3. Briefly wash three times the beads with 500 μl of precooled wash/binding buffer at 300 x g, 30 sec.
  4. Centrifuge the beads at 300 x g, 4 °C.
  5. Add 1 ml of wash/binding buffer to each tube with PI-beads.
  6. Add 2 µg of the interest GST-tagged protein to the PI-beads.
  7. Incubate the GST-tagged protein or protein domains with the beads rotating for 2 h at room temperature. For some protein domains longer incubation time or overnight 4 °C incubation may be required, depending on the affinity of the selected domain for the phosphoinositide.
  8. Wash the beads by pelleting them by centrifugation (300-400 x g at 4 °C). Then add 1 ml of wash buffer and resuspend the beads. Invert the tube 5 times and centrifuge at 4 °C, 300-400 x g. Aspirate carefully the supernatant, without touching the beads with the tip of the needle. Repeat this process 5 times.
  9. After last wash, dry the beads by aspiration and immediately add 100 μl of 2x Laemli buffer.
  10. Boil the sample at 95 °C for 5-10 min.
  11. The sample now can be kept at -20 °C upon analysis in SDS-PAGE.
  12. Centrifuge the samples 5 min at 27,000 x g before loading in SDS-PAGE gel. This will pellet the beads.
  13. The binding can be analyzed by western-blot, detecting the interest protein or domain by anti-GST antibody. GST control alone should not produce any kind of binding.

Recipes

  1. Wash/binding buffer
    10 mM HEPES (pH 7.4)
    150 mM NaCl
    0.25 % Igepal CA630
  2. 2x Reducing* Laemmli Sample Buffer
    4% SDS
    20% Glycerol
    120 mM Tris-HCl (pH 6.8)
    5% β-mercaptoethanol
    0.2% Bromophenol blue
    *It is important that the buffer is Reducing and fresh, because the reducing agent together with the boiling will be necessary for eluting the protein from the phosphoinositides beads.

Acknowledgments

This protocol has been adapted from Galvez-Santisteban et al. (2012).

References

  1. Di Paolo, G. and De Camilli, P. (2006). Phosphoinositides in cell regulation and membrane dynamics. Nature 443(7112): 651-657.
  2. Galvez-Santisteban, M., Rodriguez-Fraticelli, A. E., Bryant, D. M., Vergarajauregui, S., Yasuda, T., Banon-Rodriguez, I., Bernascone, I., Datta, A., Spivak, N., Young, K., Slim, C. L., Brakeman, P. R., Fukuda, M., Mostov, K. E. and Martin-Belmonte, F. (2012). Synaptotagmin-like proteins control the formation of a single apical membrane domain in epithelial cells. Nat Cell Biol 14(8): 838-849.
  3. Martin-Belmonte, F. and Mostov, K. (2007). Phosphoinositides control epithelial development. Cell Cycle 6(16): 1957-1961.
  4. Szentpetery, Z., Varnai, P. and Balla, T. (2010). Acute manipulation of Golgi phosphoinositides to assess their importance in cellular trafficking and signaling. Proc Natl Acad Sci U S A 107(18): 8225-8230.

简介

由Echelon Biosciences(美国盐湖城)开发的PI包被珠测定法或“PIP-Beads”是一种快速测定法,用于以定量的方式识别哪些PI能够结合给定的蛋白质结构域。它比脂质体快得多,比PIP-条带测定更可重现。 “PIP-Beads”测定法是一种生化测定法,其基本上包括将纯化的蛋白质或蛋白质结构域与合适的PI-包被的珠粒组合孵育。洗涤后,干燥并重新悬浮样品,可以通过SDS-PAGE分离方便地进行分析。磷酸肌醇(PIs)已经被表征为细胞膜结构域的重要决定因素,例如上皮极化细胞中的顶端和基底外侧结构域(Martin-Belmonte和Mostov,2007),控制膜转运(Szentpetery et al。,2010)或确定神经元突触前或突触后终点,其他功能(Di Paolo和De Camilli,2006)。这些富含磷酸肌醇的膜将蛋白质组学机制结合在一起,赋予这些膜不同的身份和功能。在许多情况下,这种蛋白质 - PIs相互作用涉及特定蛋白质膜结构域与某些PI的直接结合。这些区域中的一些被表征为诸如来自磷脂酶-C或类突触蛋白样C2结构域的PH结构域(Galvez-Santisteban等,2012),而其中一些不是。为了确定哪个PI与给定的蛋白质结构域结合,重要的是进行快速有效的测定。脂质体结合测定非常好地建立结合的动力学性质,但是它们是昂贵的并且仅允许每个实验测试几个PI。另一方面,基于PIP-带(磷脂酰 - 肌醇 - 磷酸酯)的分析是容易和快速的,然而,PI呈现在平坦的表面中,并且重现性有时是有限的。

材料和试剂

  1. PIP-珠(Echelon)与相应的感兴趣的磷酸肌醇。 Echelon根据需要提供广泛的PI和其他磷脂包被的珠子,包括一个样品包,它有广泛的 设备


    1. 冷冻离心机
    2. 振动或旋转振动器
    3. 抽吸泵
    4. 针0.7×30毫米
    5. 1.5 ml试管

    程序

    1. 开始之前
      一个。 准备或解冻纯化的GST蛋白 b。 预冷离心机。
      C。 准备洗涤/结合缓冲液。
      d。 通过用钳子或类似工具切割针尖来制造抽吸尖端(图1)。 这将使针直径最小化。


      图1.吸引提示

    2. 每个蛋白使用50微升的PI珠。 梯形提供悬浮在缓冲液50/50中的珠。
    3. 用500μl预冷的洗涤/结合缓冲液在300×g,30秒下短暂洗涤三次珠子。
    4. 将珠子在300×g,4℃下离心。
    5. 向每个管中加入1ml洗涤/结合缓冲液与PI珠。
    6. 添加2微克利息GST标记蛋白的PI珠。
    7. 孵育GST标记蛋白或蛋白域与珠在室温下旋转2小时。 对于一些蛋白质结构域,可能需要更长的孵育时间或过夜4℃的温育,这取决于所选结构域对磷酸肌醇的亲和力。
    8. 通过离心(300-400×g 在4℃下)沉淀珠粒来洗涤珠子。 然后加入1ml洗涤缓冲液,并重悬细胞。 将管反转5次,并在4℃,300-400×g离心。 小心吸出上清液,不要用针尖接触珠子。 重复此过程5次。
    9. 最后一次洗涤后,通过吸出干珠,立即加入100μl的2×Laemli缓冲液。
    10. 将样品在95℃煮沸5-10分钟。
    11. 现在,样品在SDS-PAGE中分析时可以保持在-20℃。
    12. 在装载到SDS-PAGE凝胶中之前,以27,000×g离心样品5分钟。 这将球粒。
    13. 可以通过western印迹分析结合,通过抗GST抗体检测感兴趣的蛋白质或结构域。 单独的GST控制不应产生任何种类的结合。

    食谱

    1. 洗涤/结合缓冲液
      10mM HEPES(pH7.4) 150mM NaCl 0.25%Igepal CA630
    2. 2x Reducing * Laemmli Sample Buffer
      4%SDS
      20%甘油
      120 mM Tris-HCl(pH 6.8)
      5%β-巯基乙醇 0.2%溴酚蓝
      *重要的是缓冲液是还原和新鲜的,因为还原剂连同沸腾对于从磷酸肌醇珠洗脱蛋白质是必需的。

    致谢

    该协议改编自Galvez-Santisteban等人(2012)。

    参考文献

    1. Di Paolo,G.and De Camilli,P。(2006)。磷酸肌醇在细胞调节和膜中 动态。 443(7112):651-657。
    2. Galvez-Santisteban,M.,Rodriguez-Fraticelli,AE,Bryant,DM,Vergarajauregui,S.,Yasuda,T.,Banon-Rodriguez,I.,Bernascone,I.,Datta,A.,Spivak, ,K.,Slim,CL,Brakeman,PR,Fukuda,M.,Mostov,KE和Martin-Belmonte,F。(2012)。 突触结合蛋白样蛋白控制上皮细胞中单个顶端膜结构域的形成。 Nat Cell Biol 14(8):838-849
    3. Martin-Belmonte,F。和Mostov,K。(2007)。 磷酸肌醇控制上皮发育。 细胞周期 6(16 ):1957-1961。
    4. Szentpetery,Z.,Varnai,P。和Balla,T。(2010)。 急性操作高尔基磷酸肌醇,以评估其在细胞运输和信号传导中的重要性。 > Proc Natl Acad Sci USA 107(18):8225-8230。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gálvez-Santisteban, M., Rodriguez-Fraticelli, A. E. and Martin-Belmonte, F. (2014). Phosphoinositides Coated Beads Binding Assay. Bio-protocol 4(3): e1039. DOI: 10.21769/BioProtoc.1039.
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