The AE765, AK423, AK692, AO233 and AO234 antibodies recognize human actin by immunofluorescence in HEK cells (original) (raw)

Introduction

Actin is one of the most abundant proteins in eukaryotic cells, and a major structural component of the cytoskeleton, forming networks of microfilaments in the cytoplasm of cells. The human genome contains six genes for actin (three for α-actin, one for β-actin, and two for γ-actin) (Pollard, 2016). Five recombinant antibodies (AE765, AK423, AK692, AO233 and AO234) were tested for their ability to label actin by immunofluorescence.

Materials & Methods

Antibodies: ABCD_AE765, ABCD_AK423, ABCD_AK692, ABCD_AO233 and ABCD_AO234 antibodies (http://web.expasy.org/abcd/, ABCD nomenclature) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding scFv fused to a rabbit IgG Fc. The synthesized scFv or VHH sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)3 (Table 1). HEK293 suspension cells (growing in HEK TF medium, Xell 861-0001, supplemented with 0.1% Pluronic F68, Sigma P1300) were transiently transfected with the vector coding for the scFv-Fc or VHH-Fc. Supernatants (see Table 1 for individual yields) were collected after 4 days. The antibody AJ519, which recognizes the TAC antigen (human IL2RA, Uniprot P01589), was used as a negative control (Arsimoles et al., 2020). As positive control, a commercial anti-beta actin antibody (clone 2D4H5, Proteintech 66009-1-Ig), raised against human ACTB (Uniprot P60709), was used.

Table 1. Clone number, epitope, reference and production yields for the antibodies used in this study.

Anti gen: HEK cells were cultured on glass coverslips (Menzel-Gläser, 22x22 mm) and grown in DMEM GlutaMAXTM (Gibco 31966) supplemented with 8% Fetal Bovine Serum (Gibco 10270).

Protocol: The whole procedure was carried out at room temperature. Cells were rinsed once with PBS, and fixed either with (i) PBS + 4% paraformaldehyde (PAF) (w/v) (Applichem A3013) for 30 min, blocked with PBS + 40 mM ammonium chloride (NH4Cl) (Applichem A3661) for 5 min, and then permeabilized in PBS + 0.2% saponin for 5 min; or (ii) methanol at -20 oC for 2 min. Fixed cells were washed once (5 min) in PBS and once with PBS + 0.2% (w/v) BSA (PBS-BSA), and incubated for 30 min with the primary antibodies (for the ABCD antibodies, final concentration 5 mg/L in PBS-Tween; for the Proteintech antibody, 0.05 mg/L). After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min with secondary goat anti-rabbit IgG conjugated to AlexaFluor-647 (1:400, Molecular Probes A21245, for the ABCD antibodies) or anti-mouse IgG conjugated to AlexaFluor-647 (1:400, Molecular Probes A21235, for the Proteintech antibody). After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min with Phalloidin-TRITC (1 µg/ml in PBS-BSA, Sigma P1951). After 3 washes (10 min) with PBS-BSA, cells 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

Fluorescent phalloidin was used as a marker to label polymerized actin filaments in PAF-fixed HEK cells (Fig. 1, in white). Four anti-actin antibodies, AE765, AK692, AO233 and AO234, label some of the same structures stained with phalloidin (Fig. 1, in cyan; zoomed-in regions, identified by arrows, can be seen in Fig. 2). They also label the cellular cytoplasm (Fig. 2, pinheads). Although this cytosolic staining may represent a background staining, it seems more likely that it is due to detection of non-polymerized actin. No staining was observed when the primary anti-TAC antibody was used as a negative control (Fig. 1, ‘Neg ctr’). In PAF-fixed HEK cells, no staining was observed with the commercial anti-actin 2D4H5 antibody, nor with AK423 (Figs. 1 and 2).

Figure 1. Actin labelling in PAF-fixed HEK cells. Labeling with AE765, AK692, AO233 and AO234 antibodies (‘anti-Actin’, in cyan) partially co-localizes with phalloidin staining (‘Phalloidin’, in white). Labeling of cytoplasmic structures not labelled by phalloidin was also seen. No labelling was observed when an unrelated primary antibody (anti-TAC AJ519) was used (‘Neg ctr’ panel). No labelling was seen with AK423 or the commercial 2D4H5 antibodies. Scale bar: 20 µm.

Figure 2. Actin labelling in PAF-fixed HEK cells. Insets from Fig. 1, showing partial co-localization of AE765, AK692, AO233 and AO234 with phalloidin (arrows) and the labelling of cytoplasmic structures not labelled by phalloidin (pinheads). Scale bar: 10 µm.

Conflict of interest

The authors declare no conflict of interest.