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Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites
PF13_0191-GFP 表达寄生虫的免疫透射电子显微分析   

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参见作者原研究论文

本实验方案简略版
PLOS Pathogens
Aug 2013

Abstract

This protocol was used to prepare pre-embedding samples of Plasmodium falciparum blood stage parasites that overexpressed the parasite protein PF13_0191 tagged with GFP. Using GFP-specific antibodies and Protein A-Gold the localisation of the overexpressed protein in the infected host cell was determined using standard transmission electron microscopy (EM). Pre-embedding EM is a common method where the antibodies are introduced before embedding and sectioning. This method avoids the problem that antigens are often difficult to detect on EM-sections after embedding. In the method described here antigens in the parasite-infected host cell are detected. Entry of the antibody is made possible through permeabilisation of the host cell with tetanolysin. In principle this method could also be used to detect antigens within the parasite if the sample is appropriately fixed and permeabilised before addition of the relevant antibody. While access of the antibody will avoid the detection problems often seen with post-embedding methods, this procedure will produce comparably poorer morphology.

Keywords: Parasitology (parasitology), Malaria (疟疾), Plasmodium falciparum (恶性疟原虫), Electron Microscopy (电子显微镜)

Materials and Reagents

  1. Parasite culture
    1. Plasmodium falciparum wildtype 3D7 parasites transfected with your protein of interest tagged with GFP
    2. Sterile, human 0+ erythrocyte concentrate (Blood Banking)
    3. RPMI complete medium (see Recipes)
      1. RPMI-1640 (AppliChem GmbH, catalog number: A1538,9010 )
      2. NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
      3. Glucose (Merck KgaA, catalog number: 1.08342.1000 )
      4. Albumax II (Life Technologies, Gibco®, catalog number: 11021-037 )
      5. Hypoxanthine (Sigma-Aldrich, catalog number: H9636 )
      6. Gentamicin (Ratiopharm GmbH)

  2. Pre-embedding samples
    1. Percoll (GE Healthcare, catalog number: 17-0891-02 )
    2. Formaldehyde (Polysciences)
    3. Tetanolysin (List Biological Labs, catalog number: 199 )
    4. BSA (Enzo Life Sciences, Biomol®)
    5. Mouse anti-GFP (Roche Diagnostics, catalog number: 11814460001 )
    6. Rabbit anti-mouse linker antibodies (Dako, catalog number: Z0259 )
    7. Protein A-Gold (6 nm) (Aurion, catalog number: 806-111 )
    8. 25% Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16210 )
    9. OsO4 (Electron Microscopy Sciences, catalog number: 19152 )
    10. Low melting agarose (Electron Microscopy Sciences, catalog number: 10207 )
    11. 100% ethanol (Merck KGaA)
    12. Propylene oxide (Science Services GmbH, catalog number: UN1280 )
    13. Gelantine capsules (Plano, catalog number: G29212 )
    14. Uranyl acetate (Agar Scientific, catalog number: R1260 )
    15. Methyl nadic anhydride (MNA) (Agar Scientific, catalog number: R1083 )
    16. Dodecenylsuccinic anhydride (DDSA) (Agar Scientific, catalog number: R1053 )
    17. EPON (Agar Scientific, catalog number: R1045 )
    18. Benzyldimethylamine (BDMA) (Agar Scientific, catalog number: R1062b )
    19. Pb(NO3)2 (Merck KGaA, catalog number: 12438 )
    20. Na(CH3)2AsO2.3H2O (Fluca)
    21. Saccharose (Merck KGaA)
    22. 10x PBS (see Recipes)
    23. Tetanolysin (see Recipes)
    24. Sodium cacodylate buffer (see Recipes)
    25. EPON (see Recipes)
    26. Lead citrate (pH 12) (Reynolds' stain) (see Recipes)

Equipment

  1. Falcon tubes (15 ml, 50 ml)
  2. Centrifuge
  3. Eppendorf tubes (1.5 ml, 2 ml)
  4. Sterilisation filters (0.22 µm)
  5. Bunsen burner
  6. Water bath
  7. Glass bottles
  8. Thermo block
  9. Rolling device
  10. Razor blades
  11. Sealable glass vials
  12. Tweezers
  13. Ultramicrotome (Leica Microsystems)
  14. Copper grids (Plano)
  15. Transmission electron microscope (FEI)

Procedure

  1. Preparation of pre-embedding samples
    1. Centrifuge 4 x 10 ml of a Plasmodium falciparum culture (5-10% trophozoites and schizonts) in 15 ml falcon tubes at 500 x g for 5 min, discard the supernatant and resuspend the pellet in 4 x 10 ml 1 x PBS. Centrifuge again.
    2. Purify infected red blood cells from culture using a Percoll gradient as described in Bio-protocol Immuno-EM analysis of PF13_0191-GFP Expressing Parasites (steps A2a-e) (Heiber and Spielmann, 2014).
      Note: Combine the 4 pellets after the first washing step into one 1.5 ml Epppendorf tube.
    3. Resuspend the pellet in 1 ml 1x PBS containing 2% formaldehyde and incubate at room temperature for 10 min on a rolling device.
    4. Centrifuge at 300 x g for 3 min and wash the pellet 3x in 1 ml 1x PBS.
    5. Resuspend the pellet in 800 µl 1x PBS, add 8 µl tetanolysin (1 µg/µl) and mix the solution immediately by flipping the tube with your finger. Incubate at 37 °C for 30 min and prevent the cells from sinking to the bottom by flipping the tube with your finger every few minutes.
    6. Centrifuge at 300 x g for 3 min and wash the pellet once by resuspending it in 1 ml 1x PBS followed by centrifuge at 300 x g for 3 min.
    7. Resuspend the pellet again in 1 ml 1x PBS containing 2% formaldehyde and incubate at room temperature for 5 min on a rolling device.
    8. Centrifuge at 300 x g for 3 min and wash the pellet once by resuspending it in 1 ml 1x PBS followed by centrifuge at 300 x g for 3 min.
    9. Resuspend the pellet in 500 µl 1x PBS containing 3% BSA and incubate at room temperature for 15 min on a rolling device.
    10. Centrifuge at 300 x g for 3 min. Resuspend the pellet in 200 µl of freshly prepared 1:20 dilution of mouse anti-GFP antibody in 3% BSA in 1x PBS and incubate at room temperature for 1.5 h on a rolling device.
    11. Centrifuge at 300 x g for 3 min and wash the pellet twice in 1 ml 1x PBS.
    12. Resuspend the pellet in 200 µl of freshly prepared 1:25 dilution of rabbit anti-mouse linker antibody in 3% BSA in 1x PBS and incubate at room temperature for 1.5 h on a rolling device.
    13. Centrifuge at 300 x g for 3 min and wash the pellet twice in 1 ml 1x PBS.
    14. Resuspend the pellet in 200 µl of freshly prepared 1:20 dilution of protein A gold in 3% BSA in 1x PBS and incubate at room temperature for 1.5 h on a rolling device.
    15. Centrifuge at 300 x g for 3 min and wash the pellet twice in 1 ml 1x PBS.

  2. Fixation
    1. Resuspend the cells in 500 µl sodium cacodylate buffer and add glutaraldehyde to 2% and incubate for 1 h at 4 °C.
      Note: The sample can be stored at 4 °C until use.
    2. Centrifuge at 6,000 x g for 3 min and wash the cells 3x with 500 µl sodium cacodylate buffer.
    3. Post fix the cells with 1% OsO4 in cacodylate buffer for 30 min at 4 °C.
    4. Centrifuge at 6,000 x g for 3 min and wash the cells 3x with 500 µl sodium cacodylate buffer.
    5. Prepare 3% low melting agarose by heating it over a Bunsen burner and cool down to 40 °C in a water bath.
    6. Resuspend the cells in the 40 °C liquid 3% low melting agarose and in a 1.5 ml Eppendorf tube spin the cells down immediately at 13,000 x g for 5 min. Let the agarose harden for 30 min at 4 °C to form a stable agarose pellet containing the infected red blood cells.
    7. Crop off the tip of the tube with a razor blade and remove the agarose pellet.

  3. Dehydration of the sample through increasing concentrations of ethanol
    1. Start with 30% ethanol and incubate for 10 min. Continue by transferring the sample to glass bottles containing 50%, 70%, 80%, 90%, respectively and 3x to a new bottle with 100% ethanol and incubate each time for 10 min.
    2. Remove the agarose pellet from the ethanol and trim the embedded infected red blood cells with a razor blade into very small pieces (max. length of the edge ~1 mm).
    3. Incubate the sample 2x for 10 min in propylene oxide to fully cover the sample.

  4. Embedding and sectioning
    Note: All steps have to be performed in glass vials since the propylene oxide dissolves the plastic tubes and sufficient volumes need to be used to fully cover the sample.
    1. Incubate the sample in 3:1 propylene oxide/epon resin for 15 min in a sealed glass vial.
    2. Incubate the sample in 1:1 propylene oxide/epon resin for 30 min in a sealed glass vial.
    3. Incubate the sample in 1:3 propylene oxide/epon resin overnight in a sealed glass vial.
    4. Replace the resin mixture with fresh pure epon resin and incubate in an unsealed glass vial for 2 h at room temperature.
    5. Incubate for ~15 min at 60 °C to increase the fluidity of the resin.
    6. Fill gelatine capsules with resin and add the sample with tweezers. Incubate the samples over night at 60 °C to harden the resin.
      Note: The sample will sink to the bottom of the capsule.
    7. Prepare 50 to 70 nm ultrathin sections with an ultramicrotome and place them on copper grids using tweezers.
    8. Contrast the thin sections by placing them on a drop of 2% uranyl acetate for 5 min, followed by thorough rinsing in dH2O. Then place sections on a drop of lead citrate for 5 min, rinse thoroughly in dH2O and view the samples in a transmission electron microscope at 80 kV. An example for the appearance of infected RBCs in a section prepared according to the pre-embedding method is shown in Figure 1.


    Figure 1. Example of a section of the pre-embedded parasite material. Size bar 2 µm

Recipes

  1. RPMI complete medium (1 L)
    15.87 g RPMI-1640
    1 g NaHCO3
    2 g glucose
    5 g Albumax II
    0.0272 g hypoxanthine
    0.5 ml gentamicin (40 mg/ml)
    Adjust pH to 7.2 using HCl, 1 L dH2O
    Filter sterilize (0.22 µm) and stored at 4 °C
  2. 10x PBS (1 L)
    5.7 g Na2HPO4
    1.25 g NaH2PO4
    15.2 g NaCl
    Adjust pH to pH 7.4, 1 L dH2O
  3. Tetanolysin
    Dissolved at 1 µg/µl in dH2O
    Make aliquots and store at -20 °C
  4. Sodium cacodylate buffer (0.5 L, 0.1 M, pH 7.2)
    10.7 g Na(CH3)2AsO2.3H2O
    17.12 g saccharose
    0.111 g CaCl2
    460 ml dH2O
    40 ml 0.1 M HCl
  5. EPON
    13.4 g MNA
    10.78 g DDSA
    25.82 g EPON
    Agitate for 30 min
    Add 1.5% BDMA
    Agitate for 5 min
    Stored at -20 °C
  6. Lead citrate (pH 12) (Reynolds' stain)
    1.33 g Pb(NO3)2
    1.76 g tri-Natriumcitrate.2H2O
    17.12 g saccharose
    30 ml H2O
    Agitate for 1 min and incubate for 30 min
    Add 8 ml NaOH (Solution has to be clear.)
    Add H2O to 50 ml and stored in a sealed glass container (The solution is stable for 6 months.)
    For use dilute 1:10 in 0.1 M NaOH

Acknowledgments

We gratefully acknowledge the work from the Tilley lab that developed the method for pre-embedding EM in selectively permeabilsed P. falciparum-infected cells that forms the basis for the procedures described here: Jackson et al. (2007). The Percoll gradient is a modified version of the protocol from Aley et al. (1986). The Percoll purification is described in more detail in the Bio-protocol 'Preparation of parasite protein extracts and Western blot analysis' (Procedure I.2.a-e).

References

  1. Aley, S. B., Sherwood, J. A., Marsh, K., Eidelman, O. and Howard, R. J. (1986). Identification of isolate-specific proteins on sorbitol-enriched Plasmodium falciparum infected erythrocytes from Gambian patients. Parasitology 92 ( Pt 3): 511-525.
  2. Heiber, A. and Spielmann, T. (2014). Preparation of parasite protein extracts and western blot analysis. Bio-protocol 4(11): e1136.
  3. Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., Kadekoppala, M., Hecht, L., Holder, A. A., Gilberger, T. W. and Spielmann, T. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.
  4. Jackson, K. E., Spielmann, T., Hanssen, E., Adisa, A., Separovic, F., Dixon, M. W., Trenholme, K. R., Hawthorne, P. L., Gardiner, D. L., Gilberger, T. and Tilley, L. (2007). Selective permeabilization of the host cell membrane of Plasmodium falciparum-infected red blood cells with streptolysin O and equinatoxin II. Biochem J 403(1): 167-175.

简介

该方案用于制备过表达用GFP标记的寄生虫蛋白PF13_0191的恶性疟原虫血液阶段寄生虫的预嵌入样品。使用GFP特异性抗体和蛋白A-Gold,使用标准透射电子显微镜(EM)测定过表达的蛋白在感染的宿主细胞中的定位。预嵌入EM是其中在包埋和切片之前引入抗体的常见方法。该方法避免了嵌入后在EM-切片上通常难以检测抗原的问题。在本文所述的方法中,检测寄生虫感染的宿主细胞中的抗原。通过宿主细胞用破伤风溶蛋白透化可以进入抗体。原则上,如果样品在添加相关抗体之前被适当地固定和透化,则该方法也可以用于检测寄生虫内的抗原。虽然抗体的获取将避免嵌入后方法中经常见到的检测问题,但是该方法将产生相对较差的形态。

关键字:parasitology, 疟疾, 恶性疟原虫, 电子显微镜

材料和试剂

  1. 寄生虫培养
    1. 用用GFP标记的感兴趣蛋白质转染的恶性疟原虫野生型3D7寄生虫
    2. 无菌,人类0 + 红细胞浓缩物(Blood Banking)
    3. RPMI完全培养基(见配方)
      1. RPMI-1640(AppliChem GmbH,目录号:A1538,9010)
      2. NaHCO 3(Sigma-Aldrich,目录号:S5761)
      3. 葡萄糖(Merck KgaA,目录号:1.08342.1000)
      4. Albumax II(Life Technologies,Gibco ,目录号:11021-037)
      5. 次黄嘌呤(Sigma-Aldrich,目录号:H9636)
      6. 庆大霉素(Ratiopharm GmbH)

  2. NaHCO 3(Sigma-Aldrich,目录号:S5761)
  3. 葡萄糖(Merck KgaA,目录号:1.08342.1000)
  4. Albumax II(Life Technologies,Gibco ,目录号:11021-037)
  5. 次黄嘌呤(Sigma-Aldrich,目录号:H9636)
  6. 庆大霉素(Ratiopharm GmbH)

...
  • Rabbit anti-mouse linker antibodies (Dako, catalog number: Z0259)
  • Protein A-Gold (6 nm) (Aurion, catalog number: 806-111)
  • 25% Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16210)
  • OsO4 (Electron Microscopy Sciences, catalog number: 19152)
  • Low melting agarose (Electron Microscopy Sciences, catalog number: 10207)
  • 100% ethanol (Merck KGaA)
  • Propylene oxide (Science Services GmbH, catalog number: UN1280)
  • Gelantine capsules (Plano, catalog number: G29212)
  • Uranyl acetate (Agar Scientific, catalog number: R1260)
  • Methyl nadic anhydride (MNA) (Agar Scientific, catalog number: R1083)
  • Dodecenylsuccinic anhydride (DDSA) (Agar Scientific, catalog number: R1053)
  • EPON (Agar Scientific, catalog number: R1045)
  • Benzyldimethylamine (BDMA) (Agar Scientific, catalog number: R1062b)
  • Pb(NO 3)2(Merck KGaA,目录号:12438)
  • Na(CH 3)2 Sub 2 O 2(Fluca)3 H 2 O 2(CH 3)
  • Saccharose(Merck KGaA)
  • 10x PBS(请参阅配方)
  • 溶栓素(见配方)
  • 二甲胂酸钠缓冲液(见配方)
  • EPON(参见配方)
  • 柠檬酸铅(pH 12)(雷诺氏染色)(见配方)
  • 设备

    1. Falcon管(15ml,50ml)
    2. 离心机
    3. Eppendorf管(1.5ml,2ml)
    4. 灭菌过滤器(0.22μm)
    5. 本生灶
    6. 水浴
    7. 玻璃瓶
    8. 热块
    9. 滚动装置
    10. 剃刀刀片
    11. 可密封的玻璃小瓶
    12. 镊子
    13. 超薄切片机(Leica Microsystems)
    14. 铜网(Plano)
    15. 透射电子显微镜(FEI)

    程序

    1. 预嵌入样品的制备
      1. 离心4×10ml镰状疟原虫培养物(5-10% 滋养体和裂殖体)在15ml falcon管中以500×g离心5分钟, 弃去上清液并将沉淀物重悬于4×10ml 1×PBS中。 再次离心。
      2. 使用Percoll梯度从培养物中纯化感染的红细胞,如生物方案的 PF13_0191-GFP的免疫-EM分析 表示寄生虫(步骤A2a-e)(Heiber和Spielmann,2014) 注意:在第一次洗涤步骤后,将4个丸粒合并到一个1.5 ml Epppendorf管中。
      3. 将沉淀重悬于含有2%甲醛的1ml 1×PBS中 在滚动装置上在室温下孵育10分钟
      4. 在300×g离心3分钟,并在1ml 1×PBS中洗涤沉淀3次。
      5. 重悬沉淀在800μl1×PBS,加入8μl溶组蛋白溶酶(1μg/μl) 并用手指翻转管子立即混合溶液。 在37℃孵育30分钟,并防止细胞下沉 底部,每隔几分钟用手指翻转管
      6. 在300×g离心3分钟并洗涤沉淀一次 将其重悬于1ml 1×PBS中,然后以300×g离心3分钟 min。
      7. 再次悬浮在1ml 1×PBS含有2% 甲醛并在室温下温育5分钟 设备。
      8. 在300×g离心3分钟并洗涤沉淀 一次通过将其重悬浮在1ml 1x PBS中,随后在300×g离心3分钟。
      9. 将沉淀重悬在500μl含有3%BSA的1×PBS中,并在滚动装置上在室温下孵育15分钟。
      10. 在300×g离心3分钟。 重悬在200μl的沉淀 新鲜制备的1:20稀释的在3%BSA中的小鼠抗GFP抗体 1x PBS,并在滚动装置上在室温下孵育1.5小时
      11. 在300×g离心3分钟,并在1ml 1×PBS中洗涤沉淀两次。
      12. 将沉淀重悬于200μl新鲜制备的1:25稀释液中 兔抗小鼠接头抗体在1%PBS中的3%BSA中, 室温在滚动装置上保持1.5小时
      13. 在300×g离心3分钟,并在1ml 1×PBS中洗涤沉淀两次。
      14. 将沉淀重悬于200μl新鲜制备的1:20稀释的 蛋白A金在1%PBS中的3%BSA中并在室温下孵育   在滚动装置上为1.5小时
      15. 在300×g离心3分钟,并在1ml 1×PBS中洗涤沉淀两次。

    2. 固定
      1. 重悬细胞在500微升的二甲胂酸钠缓冲液,添加戊二醛至2%,并在4℃孵育1小时。
        注意:样品可以在4°C储存,直到使用。
      2. 以6000xg离心3分钟,并用500μl二甲胂酸钠缓冲液洗涤细胞3次。
      3. 在4℃下用1%OsO 4在二甲胂酸盐缓冲液中将细胞后固定30分钟。
      4. 以6000xg离心3分钟,并用500μl二甲胂酸钠缓冲液洗涤细胞3次。
      5. 通过在本生灯上加热制备3%低熔点琼脂糖,并在水浴中冷却至40℃。
      6. 重悬细胞在40℃液体3%低熔点琼脂糖和   1.5ml Eppendorf管使细胞立即以13,000×g离心   5分钟。 让琼脂糖在4℃下硬化30分钟以形成稳定 含有感染的红细胞的琼脂糖沉淀
      7. 用刀片切下管的尖端,取出琼脂糖沉淀
    3. 通过增加乙醇浓度使样品脱水
      1. 开始用30%乙醇并孵育10分钟。 继续转移   样品到玻璃瓶分别含有50%,70%,80%,90% 和3x到100%乙醇的新瓶,每次孵育10 min。
      2. 从乙醇中取出琼脂糖沉淀并修剪 嵌入的感染红细胞用剃刀刀片变得非常小 (边缘最大长度〜1 mm)。
      3. 将样品在环氧丙烷中孵育10分钟,以完全覆盖样品。

    4. 嵌入和划分
      注意:所有步骤都必须在玻璃瓶中进行,因为环氧丙烷溶解塑料管,并且需要足够的体积来完全覆盖样品。
      1. 在密封的玻璃小瓶中将样品在3:1环氧丙烷/epon树脂中孵育15分钟
      2. 在密封的玻璃小瓶中将样品在1:1环氧丙烷/epon树脂中孵育30分钟
      3. 将样品在1:3环氧丙烷/epon树脂中在密封的玻璃小瓶中孵育过夜
      4. 用新鲜纯的epon树脂替换树脂混合物,并在未密封的玻璃小瓶中在室温下孵育2小时。
      5. 在60℃下孵育〜15分钟以增加树脂的流动性。
      6. 填充明胶胶囊与树脂,并用镊子添加样品。 将样品在60℃下孵育过夜,以使树脂硬化 注意:样品将沉到胶囊底部。
      7. 使用超薄切片机制备50至70 nm超薄切片,并使用镊子将其放置在铜网格上。
      8. 通过将薄片放在2%的铀酰滴上来对比薄片 乙酸乙酯5分钟,随后在dH 2 O中彻底漂洗。 然后放置 在一滴柠檬酸铅上的切片5分钟,在dH 2 O中彻底冲洗 并在80kV下在透射电子显微镜下观察样品。 一个 在制备的切片中感染的RBC的外观的实例 根据预嵌入方法如图1所示。


      图1.预嵌入寄生虫材料的一部分示例。尺寸栏2μm

    食谱

    1. RPMI完全培养基(1L)
      15.87g RPMI-1640
      1g NaHCO 3 3/h 2 g葡萄糖 5克Albumax II
      0.0272g次黄嘌呤 0.5ml庆大霉素(40mg/ml) 使用HCl,1L dH 2 O 2调节pH至7.2 过滤灭菌(0.22μm)并在4℃下保存
    2. 10x PBS(1L)
      5.7g Na 2 HPO 4。 1.25g NaH 2 PO 4 sub/d 15.2g NaCl
      将pH调节至pH 7.4,1L dH 2 O
    3. 溶血素 在dH 2 O中溶解为1μg/μl
      将等分试样并保存在-20°C
    4. 二甲胂酸钠缓冲液(0.5L,0.1M,pH7.2) 10.7g Na(CH 3)2 sub 2 AsO 2 sub。3 H 2 O 2。 br /> 17.12克蔗糖 0.111g CaCl 2
      460ml dH 2 O
      40ml 0.1M HCl
    5. EPON
      13.4克MNA
      10.78g DDSA
      25.82克EPON
      搅拌30分钟
      添加1.5%BDMA
      搅拌5分钟
      储存于-20°C
    6. 柠檬酸铅(pH 12)(雷诺氏染色)
      1.33g Pb(NO 3)2 sub。 1.76g柠檬酸三钠。 2H 2 17.12克蔗糖 30ml H 2 O 2 / 搅拌1分钟,孵育30分钟
      加入8ml NaOH(溶液必须澄清。)
      将H 2 O加至50ml并储存在密封的玻璃容器中(该溶液稳定6个月)。
      使用时稀释1:10在0.1 M NaOH中

    致谢

    我们衷心感谢来自Tilley实验室的工作,该实验室开发了在选择性渗透的恶性疟原虫感染的细胞中预先嵌入EM的方法,其形成了本文所述的程序的基础:Jackson等。(2007)。 Percoll梯度是来自Aley等人的方案的修改版本(1986)。在Bio-protocol"寄生虫蛋白提取物的制备和蛋白质印迹分析"(方法I.2.a-e)中更详细地描述了Percoll纯化。

    参考文献

    1. Aley,S.B.,Sherwood,J.A.,Marsh,K.,Eidelman,O。和Howard,R.J。(1986)。 从冈比亚患者中鉴定山梨醇富集的恶性疟原虫感染的红细胞上的分离物特异性蛋白 。 寄生虫学 92(Pt 3):511-525。
    2. Heiber,A.和Spielmann,T。(2014)。 寄生虫蛋白提取物的制备和蛋白质印迹分析 生物方案 4 (11):e1136。
    3. Heiber,A.,Kruse,F.,Pick,C.,Gruring,C.,Flemming,S.,Oberli,A.,Schoeler,H.,Retzlaff,S.,Mesen-Ramirez,P.,Hiss,JA ,Kadekoppala,M.,Hecht,L.,Holder,AA,Gilberger,TW和Spielmann,T。(2013)。 新PNEPs的鉴定表明一个实质的非PEXEL exportome和支持恶性疟原虫蛋白输出的共同特点。 a> PLoS Pathog 9(8):e1003546。
    4. Jackson,KE,Spielmann,T。,Hanssen,E.,Adisa,A.,Separovic,F.,Dixon,MW,Trenholme,KR,Hawthorne,PL,Gardiner,DL,Gilberger,T.and Tilley, 2007)。 用链球菌溶血素O和equinatoxin II选择性透化恶性疟原虫感染的红细胞的宿主细胞膜 。 Biochem J 403(1):167-175
    • English
    • 中文翻译
    免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
    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. Heiber, A., Retzlaff, S. and Spielmann, T. (2014). Immuno-EM Analysis of PF13_0191-GFP Expressing Parasites. Bio-protocol 4(11): e1137. DOI: 10.21769/BioProtoc.1137.
    2. Heiber, A., Kruse, F., Pick, C., Gruring, C., Flemming, S., Oberli, A., Schoeler, H., Retzlaff, S., Mesen-Ramirez, P., Hiss, J. A., Kadekoppala, M., Hecht, L., Holder, A. A., Gilberger, T. W. and Spielmann, T. (2013). Identification of new PNEPs indicates a substantial non-PEXEL exportome and underpins common features in Plasmodium falciparum protein export. PLoS Pathog 9(8): e1003546.
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