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Membrane Lipid Screen to Identify Molecular Targets of Biomolecules
采用膜脂筛查法鉴定生物分子的分子靶点   

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Abstract

Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: ‘Liposome disruption assay to examine lytic properties of biomolecules’ which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).

Keywords: Membrane(膜), Liposome(脂质体), Lipid(脂质), Disruption(破碎), Lysis(裂解), Carboxyfluorescein(羧基荧光素), Leakage(渗漏), Specificity(特异性)

Background

Proteins and biomolecules with membrane disruption activities, such as pore formation or membrane fusion, may target specific lipids within membranes. Examples of lipid-specific pore-formation include Plasmodium CelTOS that depends on phosphatidic acid for pore formation, and the cholesterol dependent cytolysins (Jimah et al., 2016; Lukoyanova et al., 2016). CelTOS (cell traversal protein for ookinetes and sporozoites) is a malaria parasite protein that disrupts host cell membranes by pore formation to enable the exit of parasites from invaded host cells during cell traversal (Kariu et al., 2006; Jimah et al., 2016). Cholesterol dependent cytolysins are a large class of pore-forming proteins, including virulence factors of gram positive bacteria such as pneumolysin and listeriolysin (Lukoyanova et al., 2016). Identifying the specific lipids targeted informs the mechanism of membrane disruption that underlies biological function and role of proteins and biomolecules.

Materials and Reagents

  1. Small gel incubation tray (Santa Cruz Biotechnology, catalog number: sc-358889 )
  2. Serological pipets, 10 ml (Genesee Scientific, catalog number: 12-104 )
  3. Pipette tips
    10 µl tips (VWR, catalog number: 46620-318 )
    200 µl tips (VWR, catalog number: 53509-009 )
    1,000 µl tips (VWR, catalog number: 83007-384 )
  4. Centrifuge tubes, 50 ml (Genesee Scientific, catalog number: 21-106 )
  5. Microcentrifuge tubes, 1.7 ml (MIDSCI, catalog number: AVSS1700RA )
  6. Membrane lipid strips with dimensions 2 x 3 cm (Echelon Biosciences, catalog number: P-6002 )
  7. Protein or biomolecule of interest
  8. Tris (Gold Bio, catalog number: T-400-5 )
  9. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888-25KG )
  10. Tween 20% (Sigma-Aldrich, catalog number: P1379 )
  11. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906-500G )
  12. Primary antibody against the tagged protein or biomolecule of interest
  13. Peroxidase conjugated secondary antibody
  14. ECL Prime Western Blotting Detection Kit (GE Healthcare, catalog number: RPN2232 )
  15. Phospholipids (dissolved in chloroform, commonly used phospholipids are):
    DOPC (Avanti Polar Lipids, catalog number: 850375C )
    POPC (Avanti Polar Lipids, catalog number: 850457C )
    POPA (Avanti Polar Lipids, catalog number: 840857C )
    POPS (Avanti Polar Lipids, catalog number: 840034C )
  16. Blocking buffer (see Recipes)
  17. Wash buffer (see Recipes)

    Note: See the ‘Notes’ section for a list of materials and reagents used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).

Equipment

  1. Pipetman Classic pipets
    P10 (Gilson, catalog number: F144802 )
    P20 (Gilson, catalog number: F123600 )
    P200 (Gilson, catalog number: F123601 )
    P1000 (Gilson, catalog number: F123602 )
  2. Pipet-Aid XP Pipette controller (Drummond Scientific, catalog number: 4-000-101 )
  3. Incubator
  4. BenchRockerTM variable 2D rocker (Benchmark Scientific, catalog number: BR2000 )
  5. pH meter (Fisher Scientific, model: AccumetTM AE150, catalog number: 13-636-AE153 )
  6. Fluorescent image analyzer (Fujifilm, model: FLA-5000 )

    Note: See the ‘Notes’ section for a list of equipment used in the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ that is recommended for follow up experiments (Jimah et al., 2017).

Procedure

Screen to identify specific membrane lipid binding by biomolecules of interest

  1. Purify or obtain the biomolecule of interest.
    Note: Ensure the protein is tagged, for example with a 6-His tag.
  2. Place lipid strips in a small gel incubation tray, and block lipid strips with 10 ml blocking buffer (see Recipes) at room temperature for one hour.
    Notes:
    1. See the ‘Notes’ section for a description of the lipid component of the lipid strips.
    2. The buffer and pH used depend on the biomolecule tested. For example, acidic conditions could be used if the biomolecule functions in an acidic environment. Also, use enough buffer to cover the lipid strip.
  3. Wash three times, for five minutes each, with 10 ml wash buffer (see Recipes).
    Note: Suggestions on the duration of washing, blocking and incubation times is described in the Notes section.
  4. Incubate the membrane lipid strips with the protein or biomolecule in 10 ml blocking buffer for one hour at room temperature.
    Note: The concentration of protein used is determined empirically, and is usually at the low micromolar to nanomolar concentrations.
  5. Wash three times, for five minutes each, with 10 ml wash buffer.
  6. Incubate the membrane lipid strips with a primary antibody against the tagged protein or biomolecule in 10 ml blocking buffer for one hour at room temperature.
    Notes:
    1. The primary antibody could be against the protein tag or specific to the protein or biomolecule.
    2. The concentration of primary antibody used is specified by the manufacturer.
  7. Wash three times, for five minutes each, with 10 ml wash buffer.
  8. Incubate the membrane lipid strips with a peroxidase conjugated secondary antibody, against the primary antibody, in 10 ml blocking buffer for one hour at room temperature.
    Note: The concentration of secondary antibody used is specified by the manufacturer.
  9. Use the ECL Prime Western Blotting Detection Kit to detect the peroxidase conjugated secondary antibody.
  10. Image the chemiluminescence using a fluorescent image analyser (Figure 1).


    Figure 1. Lipid strips probed with PfCelTOS or controls. A. Lipid strip probed with PfCelTOS, followed by primary antibody to CelTOS and a secondary antibody conjugated with peroxidase for visualization. PfCelTOS specifically bound to phosphatidic acid (Jimah et al., 2016). B. Lipid strip probed with primary antibody against His-tag and a secondary antibody conjugated with peroxidase for visualization. Neither the primary or secondary antibody bound to the lipid strip.

Data analysis

  1. Screen to identify specific membrane lipid binding by biomolecules of interest:
    1. Spot intensities in each strip are normalized to the background for that strip and to the intensities in the negative control(s).
      Note: The negative control may be a no protein control, or a protein that does not bind lipids.
    2. The normalized spot intensities, from experimental replicates, are compared to determine if there is a statistical preference for specific lipids by one-way ANOVA.
      Note: Perform appropriate number of replicates for statistical analysis. An example of representative data and analysis is reported in Jimah et al., 2016.

  2. Conclusion
    The protocol described here enables the identification of specific lipids that are targeted by proteins of biomolecules for binding. In addition to binding lipid molecules, some proteins or biomolecules may disrupt membranes by targeting a specific lipid component of a membrane. A companion protocol: ‘Liposome disruption assay to test lytic properties of biomolecules’ describes how to determine and quantify the disruption of membranes by proteins or biomolecules (Jimah et al., 2017). Below is a summary of ‘Screen to identify membrane lipids targeted for membrane disruption by biomolecules’.
    Procedure: Screen to identify membrane lipids targeted for membrane disruption by biomolecules
    Notes:
    1. The identification of specific lipids targeted for binding by biomolecules, described above, will inform the lipids targeted for membrane disruption since binding precedes membrane disruption.
    2. The protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ will be applied to determine if the presence of a particular lipid within membranes is necessary for membrane disruption.
    1. Make liposomes composed of lipids that have been identified to bind the biomolecule of interest.
      Note: For example, if the biomolecule binds phosphatidic acid, liposomes composed of phosphatidylcholine and phosphatidic acid at 8:2 molar ratio may be made. The addition of phosphatidylcholine is because liposomes composed of phosphatidic acid alone are unstable. In the case of this example, it is also useful to make liposomes composed only of phosphatidylcholine to serve as a negative control.
    2. Investigate the membrane disruption activity of the biomolecule on the liposomes following the protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’.
    3. Test a range of concentrations of the biomolecule for the ability to disrupt liposomes.
      Note: Recommended concentrations are within the nanomolar and micromolar range. The biomolecule targets specific lipids if low nanomolar concentrations of the biomolecule disrupt membranes containing particular lipids. It may be possible that at high concentrations, the biomolecule nonspecifically disrupts membranes composed of other lipids.

      Data analysis: Screen to identify membrane lipids targeted for membrane disruption by biomolecules
      Note: Please refer to the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ for a detailed description of data analysis. (Jimah et al., 2017).

Notes

  1. Membrane lipid strips (Echelon Biosciences) contain the common membrane lipids: triglyceride, phosphatidylinositol, phosphatidylinositol (4)-phosphate, phosphatidylinositol (4,5)-bisphosphate, phosphatidylinositol (3,4,5)-trisphosphate, phosphatidylserine, phosphatidylethanolamine, phosphatidic acid, diacylglycerol, cholesterol, phosphatidylcholine, sphingomyelin, phosphatidylglycerol, 3-sulfogalactosylceramide and cardiolipin.
  2. Another membrane lipid strip contains additional lipids that may be of interest (Echelon Biosciences, catalog number: P-6001) contains: Lysophosphatidic acid (LPA), Lysophosphocholine (LPC), Phosphatidylinositol (PtdIns), Phosphatidylinositol (3) phosphate (PtdIns(3)P), Phosphatidylinositol (4) phosphate (PtdIns(4)P), Phosphatidylinositol (5) phosphate (PtdIns(5)P), Phosphatidylethanolamine (PE), Phosphatidylcholine (PC), Sphingosine 1-Phosphate (S1P), Phosphatidylinositol (3,4) bisphosphate (PtdIns(3,4)P2), Phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), Phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P2), Phosphatidylinositol (3,4,5) trisphosphate (PtdIns(3,4,5)P3), Phosphatidic acid (PA), Phosphatidylserine (PS).
  3. The blocking, incubation, and washing times stated here are recommendations, and may be modified based on empirical observations.
  4. Please refer to the companion protocol ‘Liposome disruption assay to examine lytic properties of biomolecules’ for a detailed description of the materials, reagents, equipment and protocol necessary for the identification of membrane lipids targeted for membrane disruption by biomolecules (Jimah et al., 2017).

Recipes

  1. Blocking buffer
    10 mM Tris pH 8.0
    150 mM NaCl
    0.1% Tween 20%
    3% BSA
  2. Wash buffer
    10 mM Tris pH 8.0
    150 mM NaCl
    0.1% Tween 20%

Acknowledgments

This work was supported by the Burroughs Wellcome Fund (to NHT) and National Institutes of Health (R56 AI080792 to NHT). This protocol was adapted from Jimah et al., 2016.

References

  1. Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
  2. Jimah, R. J., Schlesinger, H. P. and Tolia, H. N. (2017). Liposome disruption assay to examine lytic properties of biomolecules. Bio Protoc 7(15): e2433.
  3. Kariu, T., Ishino, T., Yano, K., Chinzei, Y. and Yuda, M. (2006). CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Mol Microbiol 59(5): 1369-1379.
  4. Lukoyanova, N., Hoogenboom, B. W. and Saibil, H. R. (2016). The membrane attack complex, perforin and cholesterol-dependent cytolysin superfamily of pore-forming proteins. J Cell Sci 129(11): 2125-2133.

简介

结合细胞膜并破坏细胞膜的蛋白可能靶向特定的磷脂。 这里我们描述了一种鉴定蛋白质和生物分子的脂质靶标的方案。 首先,我们描述了一个屏幕,用于识别由感兴趣的生物分子特异性结合的膜中的脂质。 第二,我们描述了一种确定膜中这些脂质的存在是否是膜破坏所必需的方法。 这里描述的方法用于确定疟疾疫苗候选人CelTOS通过特异性靶向磷脂酸来破坏细胞膜(Jimah等人,2016)。 该协议具有协同协议:“脂质体破碎测定以检查生物分子的溶解性质”,其可用于检查生物分子破坏由按照该协议确定的脂质靶标组成的膜的能力(Jimah等人 ,2017)。
【背景】具有膜破坏活性的蛋白质和生物分子,例如孔形成或膜融合,可以靶向膜内的特定脂质。脂质特异性孔隙形成的实例包括取决于磷脂酸用于孔形成的疟原虫CelTOS和胆固醇依赖性细胞溶质蛋白(Jimah等人,2016; Lukoyanova et al。,2016)。 CelTOS(卵磷脂和子孢子的细胞遍历蛋白)是一种疟疾寄生虫蛋白,其通过孔形成破坏宿主细胞膜,以使得细胞遍历过程中寄生虫免受入侵的宿主细胞的退出(Kariu等人,2006) ; Jimah等人,2016)。胆固醇依赖性细胞溶质蛋白是一类大量的造孔蛋白,包括革兰氏阳性细菌如肺炎杆菌毒素和李斯特里菌素的毒力因子(Lukoyanova等,2016)。识别特定的靶向脂质可以说明蛋白质和生物分子的生物功能和作用所构成的膜破坏机制。

关键字:膜, 脂质体, 脂质, 破碎, 裂解, 羧基荧光素, 渗漏, 特异性

材料和试剂

  1. 小凝胶培养皿(Santa Cruz Biotechnology,目录号:sc-358889)
  2. 血清学移液管,10 ml(Genesee Scientific,目录号:12-104)
  3. 移液器提示
    10μl提示(VWR,目录号:46620-318)
    200μl提示(VWR,目录号:53509-009)
    1000μl提示(VWR,目录号:83007-384)
  4. 离心管,50ml(Genesee Scientific,目录号:21-106)
  5. 微量离心管,1.7ml(MIDSCI,目录号:AVSS1700RA)
  6. 尺寸为2 x 3厘米的膜脂质条带(Echelon Biosciences,目录号:P-6002)
  7. 感兴趣的蛋白质或生物分子
  8. Tris(Gold Bio,目录号:T-400-5)
  9. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888-25KG)
  10. 吐温20%(Sigma-Aldrich,目录号:P1379)
  11. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906-500G)
  12. 针对标记蛋白或感兴趣的生物分子的一抗
  13. 过氧化物酶缀合的二抗
  14. ECL Prime Western Blotting Detection Kit(GE Healthcare,目录号:RPN2232)
  15. 磷脂(溶于氯仿,常用的磷脂):
    DOPC(Avanti Polar Lipids,目录号:850375C)
    POPC(Avanti Polar Lipids,目录号:850457C)
    POPA(Avanti Polar Lipids,目录号:840857C)
    POPS(Avanti Polar Lipids,目录号:840034C)
  16. 阻塞缓冲区(见配方)
  17. 洗涤缓冲液(见配方)

    注意:参见“注释”部分,列出了伴随方案“脂质体破碎测定以检查生物分子的溶解性质”中使用的材料和试剂,推荐用于随访实验(Jimah等,2017)。

设备

  1. Pipetman经典移液器
    P10(Gilson,目录号:F144802)
    P20(Gilson,目录号:F123600)
    P200(Gilson,目录号:F123601)
    P1000(Gilson,目录号:F123602)
  2. Pipet-Aid XP移液器控制器(Drummond Scientific,目录号:4-000-101)
  3. 孵化器
  4. BenchRocker TM 变量2D摇杆(Benchmark Scientific,目录号:BR2000)
  5. pH计(Fisher Scientific,型号:Accumet TM AE150,目录号:13-636-AE153)
  6. 荧光图像分析仪(Fujifilm,型号:FLA-5000)

    注意:请参阅“注释”部分,了解随机协议“脂质体破碎测定以检查生物分子的溶解性质”中使用的设备列表,推荐用于后续实验(Jimah等,2017)。 EM>

程序

筛选通过感兴趣的生物分子鉴定特异性膜脂质结合

  1. 净化或获得感兴趣的生物分子。
    注意:确保蛋白质被标记,例如使用6-His标签。
  2. 将脂质条置于小型凝胶培养皿中,并在室温下用10 ml封闭缓冲液(参见食谱)阻断脂质条带1小时。
    注意:

    1. 请参阅“注释”部分,了解脂质条的脂质成分
    2. 使用的缓冲液和pH取决于所测试的生物分子。例如,如果生物分子在酸性环境中起作用,则可以使用酸性条件。此外,使用足够的缓冲液来覆盖脂质条。
  3. 洗涤三次,每次5分钟,用10ml洗涤缓冲液(参见食谱)。
    注意:关于洗涤,阻塞和孵育时间的建议在注释部分中有所描述。
  4. 将膜脂质条带与蛋白质或生物分子在10ml封闭缓冲液中孵育1小时。
    注意:所用蛋白质的浓度经验确定,通常处于低微摩尔浓度至纳摩尔浓度。
  5. 洗涤三次,每次5分钟,用10ml洗涤缓冲液。
  6. 在10 ml封闭缓冲液中,将膜脂质条与标记蛋白或生物分子的一抗进行室温孵育1小时。
    注意:
    1. 第一抗体可能是针对蛋白质标签或针对蛋白质或生物分子特异的。

    2. 使用的一抗的浓度由制造商规定
  7. 洗涤三次,每次5分钟,用10ml洗涤缓冲液。
  8. 将膜脂质条与过氧化物酶缀合的第二抗体相对于第一抗体在10ml封闭缓冲液中在室温下孵育1小时。
    注意:使用的二抗的浓度由制造商指定。
  9. 使用ECL Prime Western Blotting检测试剂盒检测过氧化物酶偶联的二抗
  10. 使用荧光图像分析仪对化学发光进行成像(图1)

    图1.用PfCelTOS或对照物探测的脂质条。 A.用PfCelTOS探测的脂质条,其次是CelTOS的一级抗体和与过氧化物酶缀合的二级抗体用于可视化。 PfCelTOS特异性结合磷脂酸(Jimah等人,2016)。 B.用针对His标签的第一抗体探测的脂质条和与过氧化物酶缀合的二级抗体用于可视化。一级或二级抗体均不与脂质条结合。

数据分析

  1. 筛选通过感兴趣的生物分子鉴定特异性膜脂质结合:
    1. 每个条带中的斑点强度被归一化为该条带的背景和阴性对照中的强度。
      注意:阴性对照可能是无蛋白质对照或不结合脂质的蛋白质。
    2. 比较来自实验重复的归一化斑点强度,以确定单因素方差分析是否存在对特定脂质的统计偏好。
      注意:执行适当数量的重复进行统计分析。在Jimah等人,2016年报告了代表性数据和分析的一个例子。

  2. 结论
    这里描述的方案使得能够识别由生物分子的蛋白质靶向结合的特定脂质。除了结合脂质分子之外,一些蛋白质或生物分子可以通过靶向膜的特定脂质组分来破坏膜。伴随方案:“用于测试生物分子的裂解性质的脂质体破坏测定”描述了如何确定和定量蛋白质或生物分子对膜的破坏(Jimah等人,2017)。以下是“用于鉴定通过生物分子进行膜破坏的膜脂质的筛选”的总结 程序:用于鉴定通过生物分子进行膜破坏的膜脂质的筛选
    注意:
    1. 如上所述,通过生物分子靶向结合的特定脂质的鉴定将通知靶向膜分解的脂质,因为结合在膜破裂之前。
    2. 将应用“检测生物分子的溶解性质的脂质体破碎测定”方案,以确定膜中特定脂质的存在对于膜破裂是否必要。
    1. 使脂质体由已被鉴定为结合生物分子的脂质组成。
      注意:例如,如果生物分子结合磷脂酸,则可以以8:2的摩尔比形成由磷脂酰胆碱和磷脂酸组成的脂质体。磷脂酰胆碱的添加是因为单独由磷脂酸组成的脂质体是不稳定的。在这个例子的情况下,使仅由磷脂酰胆碱组成的脂质体用作阴性对照也是有用的。
    2. 根据方案“脂质体破碎测定法检查生物分子的溶解性质”,研究生物分子在脂质体上的膜破坏活性。
    3. 测试一系列浓度的生物分子以破坏脂质体的能力。
      注意:推荐的浓度在纳摩尔和微摩尔范围内。如果低纳摩尔浓度的生物分子破坏含有特定脂质的膜,则生物分子靶向特异性脂质。生物分子可能在高浓度时非特异性地破坏由其他脂质组成的膜。

      数据分析:用于鉴定通过生物分子进行膜破坏的膜脂质的筛选
      注意:有关数据分析的详细说明,请参阅伴随方案“脂质体破碎测定以检查生物分子的溶解性质”。 (Jimah等,2017)

笔记

  1. 膜脂质条(Echelon Biosciences)含有常见的膜脂质:甘油三酯,磷脂酰肌醇,磷脂酰肌醇(4) - 磷酸,磷脂酰肌醇(4,5) - 二磷酸,磷脂酰肌醇(3,4,5) - 磷酸,磷脂酰丝氨酸,磷脂酰乙醇胺,磷脂酸,二酰基甘油,胆固醇,磷脂酰胆碱,鞘磷脂,磷脂酰甘油,3-磺基半乳糖神经酰胺和心磷脂。
  2. 另一种膜脂质条包含可能感兴趣的另外的脂质(Echelon Biosciences,目录号:P-6001)包含:溶血磷脂酸(LPA),溶血磷脂胆固醇(LPC),磷脂酰肌醇(PtdIns),磷脂酰肌醇(3)磷酸酯(PtdIns )磷脂酰肌醇(4)磷酸(PtdIns(4)P),磷脂酰肌醇(5)磷酸酯(PtdIns(5)P),磷脂酰乙醇胺(PE),磷脂酰胆碱(PC),1-磷酸鞘氨醇(S1P),磷脂酰肌醇3,4)二磷酸盐(PtdIns(3,4)P 2),磷脂酰肌醇(3,5)二磷酸盐(PtdIns(3,5)P 2)),磷脂酰肌醇( 4,5)二磷酸盐(PtdIns(4,5)P 2),磷脂酰肌醇(3,4,5)三磷酸盐(PtdIns(3,4,5)P 3 ),磷脂酸(PA),磷脂酰丝氨酸(PS)
  3. 这里提到的阻塞,孵化和洗涤时间是建议,可以根据经验观察进行修改。
  4. 请参阅伴随方案“脂质体破坏测定法检查生物分子的溶解性质”,以详细描述用于鉴定通过生物分子进行膜破坏的膜脂质所需的材料,试剂,设备和方案(Jimah等人。,2017)

食谱

  1. 阻塞缓冲区
    10 mM Tris pH 8.0
    150 mM NaCl
    0.1%吐温20%
    3%BSA
  2. 洗涤缓冲液
    10 mM Tris pH 8.0
    150 mM NaCl
    0.1%吐温20%

致谢

这项工作得到了Burroughs Wellcome基金(NHT)的支持。这个协议是从2016年的Jimah等人改编而来的。

参考

  1. Jim,JR,Salinas,ND,Sala-Rabanal,M.,Jones,NG,Sibley,LD,Nichols,CG,Schlesinger,PH and Tolia,NH(2016)。< a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/27906127”target =“_ blank”>疟疾寄生虫CelTOS靶向细胞膜的内部小叶,以致孔依赖性破坏。 Elife 5.
  2. Jimah,RJ,Schlesinger,HP和Tolia,HN(2017)。< a class =“ke-insertfile”href =“http://en.bio-protocol.org/e2433?preview=true”target =“脂质体破碎测定以检查生物分子的溶解性质。生物原型7(15):e2433。
  3. Kariu,T.,Ishino,T.,Yano,K.,Chinzei,Y。和Yuda,M。(2006)。 CelTOS,一种介导向蚊子和脊椎动物宿主传播的新型疟疾蛋白质.Mol Microbiol 59(5):1369 -1379。
  4. Lukoyanova,N.,Hoogenboom,BW和Saibil,HR(2016)。造孔蛋白的膜攻击复合物,穿孔素和胆固醇依赖性细胞溶质蛋白酶超家族。细胞科学129(11):2125-2133。
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Copyright Jimah et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jimah, J. R., Schlesinger, P. H. and Tolia, N. H. (2017). Membrane Lipid Screen to Identify Molecular Targets of Biomolecules. Bio-protocol 7(15): e2427. DOI: 10.21769/BioProtoc.2427.
  2. Jimah, J. R., Salinas, N. D., Sala-Rabanal, M., Jones, N. G., Sibley, L. D., Nichols, C. G., Schlesinger, P. H. and Tolia, N. H. (2016). Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption. Elife 5.
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