RB388, RB389, RB390, RB391 and RB392 antibodies do not recognize a Dictyostelium AlyL protein by Western Blot

Abstract

The recombinant antibodies RB388, RB389, RB390, RB391 and RB392 do not detect by western blot the fulllength AlyL protein from Dictyostelium discoideum.

Introduction

AlyL (Amoeba LYsozyme Like, DDB_G0286229, UniProt #Q54M35) is a member of the amoeba lysozyme family in the amoeba D. discoideum. Here we describe the inability of five recombinant antibodies (RB388, RB389, RB390, RB391 and RB392) to detect the full-length AlyL protein by Western blot.

Materials and Methods

Antibodies: ABCD_RB388, ABCD_RB389, ABCD_RB390, ABCD_RB391 and ABCD_RB392 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) 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 Fc (MRB388, MRB389, MRB390, MRB391 and MRB392). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (~50 mg/L) were collected after 5 days. As a positive control, the anti-6xHis antibody AD946 (Lamrabet and Jauslin, 2018) was used.

Antigen: The antibodies were raised against a N-biotinylated synthetic peptide corresponding to 23 residues close to AlyL C-terminus (RVISTNVGPNV DIEDKIGKPIMD).D. discoideum DH1 (WT) cells expressing a 6xHis-tagged AlyL protein (AlyL-His, 6xHis-tag fused to the C-terminus) were used to detect the full-length AlyL protein. AlyL knockout (KO) cells were used as a negative control.

Protocol: 5x10⁶ D. discoideum cells were pelleted and resuspended in 200 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). 20 µL of each sample was migrated (200 V, 30 min) in a 10% acrylamide gel (Mini-PROTEAN® TGX™ Precast Gel, Biorad #456-1033), and transferred to a nitrocellulose membrane using a dry transfer system for 10 minutes (iBlot gel transfer device, Invitrogen #IB1001EU). The membranes were blocked during 1 hour in PBS containing 0.1% (v/v) Tween20 and 7% (w/v) milk, and washed three times for 15 minutes in PBS + 0.1% (v/v) Tween20. The membranes were then incubated with each of the six tested antibodies (dilution 1:10 and 1:2 in PBS-Tween for MRBs and AD946, respectively), overnight at 4 °C, then washed three times for 15 minutes. The membranes were then incubated with horseradish peroxidase-coupled goat anti-mouse IgG (Biorad #170-6516, dilution 1:3000) and washed twice for 15 minutes and once for 5 minutes in PBS-Tween. The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences) using a PXi-4 gel imaging systems (Syngene).

Results

Antibodies MRB388, MRB389, MRB390, MRB391 and MRB392 did not specifically recognize the AlyL protein in D. discoideum cells overexpressing a 6xHis-tagged AlyL protein (Fig. 1). The protein could be detected with an anti-6xHis antibody (AD946): a band is detected in the overexpressing cells, but is absent on alyL-KO cells.

Figure 1. 1. No specific binding of MRB antibodies to cells overexpressing AlyL-His. AlyL-His was successfully detected by the anti-6xHis AD946 antibody (position indicated by an asterisk), but not by any of the MRB antibodies.

Conflict of interest

The authors declare no conflict of interest.