RB447, RB448, RB449, RB450, RB451 and RB453 antibodies recognize a Dictyostelium AlyL protein by Western Blot

Abstract

The recombinant antibodies RB447, RB448, RB449, RB450, RB451 and RB453 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 ability of six recombinant antibodies (RB447, RB448, RB449, RB450, RB451 and RB453) to detect the full-length AlyL protein by Western blot.

Materials & Methods

Antibodies: ABCD_RB447, ABCD_RB448, ABCD_RB449, ABCD_RB450, ABCD_RB451 and ABCD_RB453 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (; Blanc et al., 2014) as mini-antibodies with the antigen-binding scFv fused to a mouse Fc (MRB447, MRB448, MRB449, MRB450, MRB451 and MRB453). 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.

Antigen: RB447 was raised against a GST protein fused to the residues 18 to 84 of the AlyL protein. RB448, RB449, RB450, and RB451 were raised against a GST protein fused to the residues 18 to 84 followed with residues 479 to 572. RB453 was raised against a GST protein fused to the C-terminal residues 534 to 572. D. discoideum alyL knockout (KO) 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 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 7.5% acrylamide gel (Mini-PROTEAN® TGX™ Precast Gel, Biorad #456-1023), 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 MRB antibodies (dilution 1:10 in PBS-Tween), overnight at 4°C, then washed three times for 15 minutes. The membranes were then incubated during 1 hour 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 MRB447, MRB448, MRB449, MRB450, MRB451 and MRB453 specifically recognize the AlyL protein in D. discoideum cells overexpressing a 6xHis-tagged AlyL protein; the protein was not detected in alyL-KO cells (Fig. 1).

Figure 1. Specific binding of MRB antibodies to cells overexpressing AlyL-His. AlyL-His was successfully detected by all six antibodies (position indicated by an asterisk). No band was observed in alyL KO cells

Conflict of interest

The authors declare no conflict of interest.