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Peer-reviewed

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

AH Arlett Heiber
SR Silke Retzlaff
TS Tobias Spielmann

发布: 2014年06月05日第4卷第11期 DOI: 10.21769/BioProtoc.1137 浏览次数: 9430

评审: Kanika GeraFanglian HeAnonymous reviewer(s)

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Cover of PLOS Pathogens, featuring study using the protocol.

Aug 2013

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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) search Malaria (疟疾) search Plasmodium falciparum (恶性疟原虫) search Electron Microscopy (电子显微镜) search

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

English
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文章信息

版权信息

© 2014 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

微生物学 > 微生物细胞生物学 > 细胞成像

生物化学 > 蛋白质 > 免疫检测

细胞生物学 > 细胞成像 > 电子显微镜

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