The AJ154 antibody recognizes the Dictyostelium p80 protein by immunofluorescence

Authors

  • Wanessa C Lima
  • Pierre Cosson

DOI:

https://doi.org/10.24450/journals/abrep.2019.e24

Abstract

The AJ154 antibody, derived from the H161 hybridoma, detects by immunofluorescence the full-length p80 protein from Dictyostelium discoideum.

Introduction

The p80 protein (DDB_G0287297, UniProt #Q7YXD4) is a widely-used marker for endosomal compartments in D. discoideum, recognized by the H161 monoclonal antibody(Ravanel et al., 2001). Here we describe the ability of the AJ154 antibody, a single chain fragment (scFv) derived from the H161 hybridoma, to label p80-endosomal compartments by immunofluorescence.

Materials & Methods

Antibodies: ABCD_AJ154 antibody (ABCD nomenclature, http://web.expasy.org/abcd/) was produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibody with the antigen-binding scFv fused to three different Fc moieties: mouse IgG2A, human IgG1 and rabbit IgG. The synthesized scFv sequence (GeneArt, Invitrogen) corresponds to the sequence of the variable regions joined by a peptide linker (GGGGS)3. The sequencing of the H161 hybridoma was performed by the Geneva Antibody Facility. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~50 mg/L) were collected after 5 days.

Antigen: D. discoideumDH1 (WT) cells were used to detect the full-length p80 protein.

Protocol: 106 D. discoideum cells were sedimented on a 22x22 mm glass coverslip (Menzel-Gläser) for 30 minutes at room temperature in HL5 medium. Cells were fixed with HL5 + 4% paraformaldehyde (w/v) (Applichem, #A3013) for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH4Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in methanol at -20 oC for 2 min, washed once (5 min) with PBS, and once (5 min) with PBS + 0.2% (w/v) BSA (PBS-BSA). Cells were then incubated for 30 min with the original mouse hybridoma H161 supernatant (dilution 1:2 in PBS-BSA) or with each of the reformatted scFv antibodies (dilution 1:10 in PBS-BSA). After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min with secondary goat anti-mouse, goat anti-rabbit or donkey anti-human IgG conjugated to AlexaFluor-488 (1:300, Molecular Probes #A11029, #A11034 and Jackson ImmunoResearch #709-545-149, respectively) or, in the case of the H161 supernatant, with goat anti-mouse IgG conjugated to AlexaFluor-647 (1:300, Molecular Probes #A21235). After 3 washes (10 min) with PBS-BSA and one wash (5 min) with PBS, coverslips were mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka, #33480). Pictures were taken using a Zeiss LSM700 confocal microscope, with a 63x Neofluar oil immersion objective.

Results

In agreement with the original description of the H161 hybridoma (Ravanel et al., 2001), the AJ154 antibody labels intracellular endocytic compartments (strongly staining post-lysosome compartments) and the cell surface (Fig. 1). The staining with AJ154 and H161 appears almost indistinguishable (Fig. 1A). Similar stainings were obtained with AJ154 exhibiting a mouse or a human Fc (Fig. 1B).

Figure 1. The H161 hybridoma and the AJ154 antibody label endosomal compartments in Dictyostelium cells. In (A), a double fluorescence staining with AJ154 and the H161 hybridoma was performed. In (B), AJ154 fused to a human or a mouse Fc shows the same labelling pattern. No labelling was seen when the primary antibody was omitted (No Ab). Scale bar: 10 µm.

Conflict of interest

Pierre Cosson and Wanessa Cristina Lima are editors of the Antibody Reports journal.

References

Ravanel K, de Chassey B, Cornillon S, et al. Membrane sorting in the endocytic and phagocytic pathway of Dictyostelium discoideum. Eur J Cell Biol. 2001;80(12):754-64. PMID:11831389

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Published

2019-03-19

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Article

How to Cite

1.
Lima WC, Cosson P. The AJ154 antibody recognizes the Dictyostelium p80 protein by immunofluorescence. Antib. Rep. [Internet]. 2019 Mar. 19 [cited 2024 May 22];2(2):e24. Available from: https://oap.unige.ch/journals/abrep/article/view/24

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