RB014, RB015 and RB016 antibodies recognize a peptide of the D. discoideum Phg1A protein by western blot

Abstract

Recombinant antibodies RB014, RB015 and RB016 detect by western blot a peptide of the Dictyostelium discoideum Phg1a protein fused to a GST protein.

Introduction

Phg1a (Phagocytic receptor 1a, DDB_G0267444, UniProt #Q55FP0) is a protein belonging to the Transmembrane 9 superfamily, involved in adhesion, phagocytosis and bacterial killing in D. discoideum (Cornillon et al., 2000). Here we describe the ability of the RB014, RB015 and RB016 antibodies to detect by western blot a fragment of the Phg1a protein fused to a GST protein.

Materials & Methods

Antibodies: ABCD_RB014, ABCD_RB015 and ABCD_RB016 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/; Lima et al., 2020) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/; Blanc et al., 2014) as mini-antibodies with the antigen-binding scFv fused to a mouse IgG2A Fc (MRB014, MRB015 and MRB016). HeLa cells (growing in DMEM GlutaMAX^TM (Gibco, #31966) supplemented with 8% Fetal Bovine Serum (Gibco, #10270)) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (~1 mg/L) were collected after 4 days.

Antigen: The antibodies were originally raised against a GST protein fused to 91 residues of the extracellular N-terminal domain of the Phg1a protein. (GEEGAIKVNKITSVHTQIPYKYYQLPGVCQPKEGIIDDTENLGEILLGDRIENSDYTFNFLTDGGKCKVINSESCSPIIKKEDLKVLEDRI).

Protocol: Expression of the GST-Phg1a recombinant protein was induced in E. coli bacteria growing exponentially (OD₆₀₀, 0.5) at 37°C (in 50 ml of Luria-Bertani (LB) medium containing 20% glucose and 100 μM ampicillin) by addition of 1.5 mM IPTG. After 3 h, bacteria were pelleted and resuspended in lysis buffer (4 ml of PBS + 1% Triton X100 + aprotinin 10 μg/ml + leupeptin 20 μg/ml + iodoacetamide 1.8 mg/ ml + PMSF 18 μg/ml) and lysed by sonication. GST was purified on glutathione-coupled sepharose 4 Fast Flow beads (GE Healthcare Life Sciences #17-5132-01), then eluted in 500 μl of reducing sample buffer (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). 15 µL of each sample was migrated (200 V, 30 min) in a 12% acrylamide gel (Mini-PROTEAN® TGX™ Precast Gel, Biorad #456-1043), and transferred to a nitrocellulose membrane using a dry transfer system for 10 minutes (iBlot gel transfer device, Invitrogen #IB1001EU). The membranes were blocked overnight at 4 °C in PBS containing 0.1% (v/v) Tween20 and 5% (w/v) milk, and washed three times (5 minutes) in PBS + 0.1% (v/v) Tween20. The membranes were then incubated with each of the tested antibodies (undiluted), for 1h at room temperature, and washed three times (5 minutes) in PBS-Tween. The membranes were then incubated with horseradish peroxidase-coupled goat anti-mouse (Biorad #170-6516, dilution 1:3000) for 1h at room temperature, and washed three times (5 minutes) in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) using a PXi-4 gel imaging systems (Syngene).

Results

RB014, RB015 and RB016 antibodies specifically recognize the GST-Phg1a fusion protein (~35 kDa), as well a higher molecular weight product at ~42 kDa. The antibodies do not bind the GST negative control (Fig. 1). The RB015 antibody also detects the full-length endogenous D. discoideum Phg1a protein by western blot (Blanc et al., 2014).

Figure 1. Specific binding of RB014, RB015 and RB016 antibodies to the GST-Phg1A protein (predicted molecular mass ~35 kDa).

Conflict of interest

The authors declare no conflict of interest.