AF394, AF395 and AF396 antibodies recognize a GFP-tagged recombinant protein by immunofluorescence

Abstract

AF394, AF395 and AF396 antibodies against the GFP protein recognize a GFP-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

Introduction

The green fluorescent protein (GFP) (Uniprot #P42212) is a large (~235 aa) protein tag, originally isolated from the jellyfish Aequorea victoria, widely used as a fluorescent reporter to detect and visualize GFP-fused proteins (Tsien, 1998). Here, we show that the AF394, AF395 and AF396 recombinant antibodies detect a GFP-tagged human TAC protein by immunofluorescence in HeLa cells.

Materials & Methods

Antibodies: ABCD_AF394,ABCD_AF396 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 nanobodies GBP1, GBP4 (for AF394 and AF395, Kirchhofer et al., 2010) and VHH (for AF396, Rothbauer et al., 2006). HEK293 suspension cells (growing in FreeStyleTM 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (50, 80, 100 mg/L for AF394, AF395 and AF396, 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 a C-terminally GFP- tagged TAC protein (Uniprot #P01589), were used to detect the protein tag. The GFP-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-GFP antibodies (final concentration 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 (1:300, Molecular Probes, #A21235). 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

AF394, AF395 and AF396 antibodies specifically detected a signal at the plasma membrane in cells transfected with the GFP-tagged TAC protein (Fig. 1). The specificity of the signal was further verified by the absence of anti-GFP staining in the few non-transfected cells (Fig. 1, arrowheads). No staining was observed when the primary antibody was omitted (Fig. 1, No Ab). The signal revealed by the antibodies mostly co-localized at the plasma membrane, with the signal generated by GFP fluorescence (Fig. 1, arrows). We note however that a GFP signal can also be detected intracellularly, and is mainly not recognized by the anti-GFP antibodies (red arrows). It is possible that part of the GFP protein is either slightly modified or not accessible to the antibodies, accounting for this observation.

Figure 1. AF394, AF395 and AF396 labeled the plasma membrane of HeLa cells expressing the GFP-tagged TAC protein (in white); the signal co-localized (arrows) with the signal generated by the GFP reporter (in green); however, intracellular GFP signal is mainly not recognized by the anti-GFP antibodies (red arrows); 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.