AE391 and AF291 antibodies recognize an HA-tagged recombinant protein by immunofluorescence

Abstract

AE391 and AF291 antibodies against the HA tag recognize an HA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

Introduction

The HA tag is a short peptide derived from the influenza virus hemagglutinin protein (Uniprot #P03435), extensively used for detection and purification of tagged proteins with anti-HA antibodies (Green at al., 1982). Here, we show that the AE391 and AF291 recombinant antibodies detect an HA-tagged human TAC protein by immunofluorescence in HeLa cells.

Materials & Methods

Antibodies: ABCD_AE391 and ABCD_AF291 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2020) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini- antibodies with the antigen-binding scFv fused to a mouse IgG2A Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions of the clones 26/9 (for AE391; Churchill et al., 1994) and 12CA5 (for AF291; Arimori et al., 2017) joined by a peptide linker (GGGS)3. HEK293 suspension cells (growing in FreeStyleTM 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (10 and 70 mg/L for AE391 and AF291, respectively) were collected after 4 days.

Antigen: HeLa cells (growing in DMEM GlutaMAXTM, Gibco #31966; supplemented with 8% Fetal Bovine Serum, Gibco #10270) cultured on glass coverslips (Menzel-Gläser, 22x22 mm) and transiently transfected 2 days before the experiment with an HA-tagged TAC protein (Uniprot #P01589), were used to detect the peptide tag. The HA epitope sequence used was YPYDVPDYASLRS and it was present in the C-terminal cytosolic domain of the fusion protein. An antibody detecting the N-terminal extracellular domain of the TAC protein (AJ519, with rabbit IgG Fc; Arsimoles et al., 2020) was used as a positive control. The HA-tagged TAC protein is expected to be mostly present at the cell surface.

Protocol: The whole procedure was carried out at room temperature. Transfected HeLa cells were rinsed once with PBS, fixed with PBS + 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 PBS + 0.2% saponin (w/v) (Sigma, #S7900) for 3 min, incubated in PBS + 0.2% (w/v) BSA (PBS-BSA) for 30 min, and then with the tested anti-HA antibodies (final concentration 5 mg/L in PBS-BSA) and AJ519 antibody (final concentration 2.5 mg/L in PBS-BSA) for 1 h. After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-mouse IgG conjugated to AlexaFluor-647 and anti-rabbit IgG conjugated to AlexaFluor-488 (1:300, Molecular Probes, #A21235 and #A11034, respectively). After 3 washes (10 min) with PBS-BSA, cells were incubated during 10 min with DAPI (1:500, Molecular Probes, #D1306), washed twice with PBS-BSA and once with PBS, and 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

AE391 and AF291 antibodies specifically detected a signal at the plasma membrane in cells transfected with the HA-tagged TAC protein (Fig. 1). The signal co-localized with the signal generated by the anti-TAC AJ519 antibody (Fig. 1, arrows); the specificity of the signal was further verified by the absence of both anti-TAC and anti-HA stainings in the few non-transfected cells (Fig. 1, arrowheads). No staining was observed when the primary antibody was omitted (Fig. 1, No Ab).

Figure 1. AE391 and AF291 labeled the plasma membrane of HeLa cells expressing the HA-tagged TAC protein (in white); the signal co- localized (arrows) with the signal generated by the anti-TAC AJ519 antibody (in green); in blue, nuclei were stained with DAPI. No labelling was seen when the primary antibody was omitted, or in non-transfected cells (arrowheads). Scale bar: 20 μm.

Conflict of interest

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