The AK423 antibody recognizes Dictyostelium and human actin with different affinities by western blot

Abstract

The AK423 antibody, raised against Dictyostelium actin, recognizes both Dictyostelium and human actin by western blot. However, it recognizes Dictyostelium actin with higher affinity than human actin.

Introduction

Actin is one of the most abundant proteins in eukaryotic cells, and a major structural component of the cytoskeleton. The AK423 antibody, originally raised against actin from the amoeba Dictyostelium discoideum (Uniprot P07830), also recognizes human actin by western blot, albeit with a lower binding affinity.

Materials & Methods

Antibodies: The ABCD_AK423 antibody (ABCD nomenclature, http://web.expasy.org/abcd/) was produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as a mini-antibody (with the antigen-binding scFv fused to a rabbit IgG Fc; Lima, 2019), and as a double-chain mouse IgG. 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 IgG. Supernatants (10 mg/L for the scFv-Fc and 30 mg/L for the IgG) were collected after 4 days. As positive control, a commercial anti-beta actin antibody (clone 2D4H5, Proteintech 66009-1-Ig), raised against human ACTB (Uniprot P60709), was used.

Antigen: HEK cells (grown in DMEM GlutaMAX^TM (Gibco 31966) supplemented with 8% Fetal Bovine Serum (Gibco 10270)) and Dictyostelium discoideum DH1 cells (cultivated in HL5 medium) were used.

Protocol: 5x10⁶ cells were pelleted and lysed for 15 min in 100 L of ice-cold lysis buffer (25 mM Tris-HCl pH 7.4 + 0.5 % Triton X-100 + 120 mM NaCl) containing protease inhibitors. Lysate was centrifuged 15 min, 10’000 g at 4 °C to remove nuclei. One volume of sample buffer was added to the lysate (20.6% (w/v) sucrose, 100 mM Tris pH 6.8, 10 mM EDTA, 0.1% (w/v) bromophenol blue, 4% (w/v) SDS, 6% (v/v) ß-mercaptoethanol) and boiled for 15 min at 95 ^oC. Dilutions of each sample (500’000, 100’000, 20’000 and 4’000 cells) were migrated (150 V, 45 min) in a 4-20% acrylamide gel (Genscript, SurePAGE Bis-Tris, M00655), and transferred to a nitrocellulose membrane using a dry transfer system for 7 minutes (iBlot gel transfer device, Invitrogen IB23001). The membranes were blocked during 60 min in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, and washed once for 15 minutes in PBS + 0.1% (v/v) Tween20 (PBS-Tween). The membranes were then incubated overnight at RT with the anti-actin antibodies (final concentration 50 or 500 ng/mL in PBS-Tween). The membranes were washed three times (15+15+10 min) in PBS-Tween, incubated for 1 hour with the horseradish peroxidase-coupled goat anti-mouse IgG (Biorad 170-6516, dilution 1:3000) or goat anti-rabbit IgG (Sigma-Aldrich A8275, dilution 1:3000) and washed three times (15 min) in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging systems (Syngene).

Results

AK423 is a recombinant antibody derived from the 224-236-1 hybridoma (Westphal et al., 1997), raised against actin from the amoeba Dictyostelium discoideum. Previously, AK423 has been described as detecting actin from Dictyostelium discoideum(Lima, 2019) as well as human actin (Marchetti, 2022), albeit at a rather high concentration (5 mg/mL). A commercial anti-beta actin antibody, used as a positive control in previous experiments, is utilized at a much lower concentration (50 ng/mL; Marchetti, 2022). We tested AK423 in two recombinant formats, as a mini-antibody (scFv fused to an Fc) and as a full IgG molecule, at the same concentration as the commercial antibody (50 ng/mL). At 50 ng/mL, AK423, in both formats, detected very efficiently Dictyostelium actin but not human actin (Fig. 1). The commercial antibody detected both Dictyostelium and human actin (Fig. 1). AK423 detected human actin only at a higher concentration (500 ng/mL) (Fig. 1). As observed before (Marchetti, 2022), AK423 also cross-reacted with a band of higher molecular weight (~130-170 kDa) in human lysates (Fig. 1).

Figure 1. The AK423 (at a final concentration of 50 ng/mL) recognizes the actin protein from Dictyostelium cells (predicted molecular mass ~42 kDa). At a higher concentration (500 ng/mL) it also recognizes human actin. The control IgG is a commercial anti-beta actin antibody from Proteintech. AK423 was tested in two different recombinant formats: as a full IgG molecule (AK423 IgG) and as a mini-antibody (AK423 scFv). For each antibody, four sample dilutions were used: 500’000, 100’000, 20’000 and 4’000 cells per well, as indicated for control antibody.

Conflict of interest

The authors declare no conflict of interest.