The AJ520 antibody recognizes the Dictyostelium vacuolar H+-ATPase subunit A by immunofluorescence

Abstract

The AJ520 antibody, derived from the 221-35-2 hybridoma, detects by immunofluorescence the full-length vacuolar H⁺-ATPase subunit A from Dictyostelium discoideum.

Introduction

The vacuolar H⁺-ATPase subunit A protein (VatA, DDB_G0287127, UniProt #P54647) is a membrane protein present in the contractile vacuole and endosomal compartments in D. discoideum, recognized by the 221-35-2 monoclonal antibody(Jenne et al., 1998; Neuhaus et al., 1998). Here we describe the ability of the AJ520 antibody, a single chain fragment (scFv) derived from the 221-35-2 hybridoma, to label the contractile vacuole and endosomes by immunofluorescence.

Materials & Methods

Antibodies: ABCD_AJ520 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 a mouse IgG2A Fc. 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 221-35-2 hybridoma was performed by the Geneva Antibody Facility. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (~50 mg/L) were collected after 5 days.

Antigen: 5x10⁵ D. discoideum DH1 cells, sedimented on a 22x22 mm glass coverslip (Menzel-Gläser) for 90 minutes at room temperature in HL5 medium, were used to detect the full-length protein.

Protocol: 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 (15 min) with PBS + 0.2% (w/v) BSA (PBS-BSA). Cells were then incubated for 30 min with the original mouse hybridoma 221-35-2 supernatant (dilution 1:3 in PBS-BSA) or with the reformatted scFv antibody (dilution 1:10 in PBS-BSA). After 3 washes (5, 5, 15 min) with PBS-BSA, cells were incubated for 30 min with secondary goat anti-mouse IgG conjugated to AlexaFluor-488 (hybridoma) or AlexaFluor-647 (scFv) (1:300, Molecular Probes #A11029 and #A21235, respectively). After 3 washes (5, 5, 15 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 descriptions of the 221-35-2 antibody(Jenne et al., 1998; Neuhaus et al., 1998), the AJ520 antibody labels the tubules and vacuoles of the contractile vacuole system and endosomal compartments (Fig. 1).

Figure 1. The 221-35-2 hybridoma and the AJ520 scFv antibody label the contractile vacuole system (pinheads) and endosomal compartments (arrowheads) in Dictyostelium cells. No labelling was seen when the primary antibody was omitted (No Ab). Scale bar: 10 µm.

Conflict of interest

The authors declare no conflict of interest.