RB155, RB156 and RB189 antibodies do not recognize the D. discoideum Tsg101 protein by western blot

Abstract

The recombinant antibodies RB155, RB156 and RB189 do not detect by western blot the full-length Tsg101 protein from Dictyostelium discoideum.

Introduction

Tsg101 (DDB_G0286797, UniProt #Q54LJ3) is a member of the ESCRT-I complex in the amoeba D. discoideum. Here we describe that three recombinant antibodies (RB155, RB156 and RB189) directed against the N-terminus of Tsg101 were not able to detect the full-length Tsg101 protein by western blot.

Materials & Methods

Antibodies: ABCD_RB155, ABCD_RB156, and ABCD_RB189 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 IgG2A Fc (MRB155, MRB156 and MRB189). 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. The rabbit polyclonal J81 antibody recognizing Tsg101 was a kind gift of Dr. L. Aubry (CEA, Grenoble, France).

Antigen: The antibodies were originally raised against a GST protein fused to the 20 first residues of Tsg101 (MYGHHGYPMHAHQQQMVNPT) This chimeric GST-Tsg101 was used as antigen for antibody selection. GST was used as negative control.

Protocol: D. discoideum cells were collected, washed in Sorensen-120 mM Sorbitol, counted and resuspended as to have 5x10⁷ cells/ml in Laemmli Buffer (125 mM Tris pH 6.8, 4% (w/v) SDS, 20% glycerol, 0.01% (w/v) bromophenol blue, 10 mM DTT). 10 µL of each sample was migrated (50 V stacking and 150 V running, 1h30) in a 10% homemade acrylamide gel and transferred to a nitrocellulose membrane (Amersham, Protran GE10600002) in 25 mM Tris, 192 mM glycine, 20% MeOH, 0.01% SDS at 4 °C, 30 V, 16 h. After checking transfer by Ponceau Red staining, the membranes were blocked during 1 hour in PBS containing 5% (w/v) BSA (bovine serum albumin fraction V, pH 7.0 (SERVA Electrophoresis GmbH 11930)). The membranes were then incubated with each of the three MRB antibodies (dilution 1:2 in PBS with or without Tween and 3% (w/v) milk) or the J81 antibody (dilution 1:500 in PBS with Tween and 3% (w/v) milk), overnight at 4 °C, then washed three times for 10 minutes in PBS. The membranes probed with the MRB antibodies were then incubated during 1 h with goat anti-mouse IgG coupled to horseradish peroxidase (Brunschwig, dilution 1:10’000 in PBS and 3% (w/v) BSA) and washed three times for 10 minutes in PBS. The membrane probed with the J81 antibody was incubated during 1 h with goat anti-rabbit coupled to horseradish peroxidase (dilution 1:10’000 in PBS-Tween and 3% (w/v) milk). The signal was revealed by enhanced chemiluminescence (ECL) (Amersham Biosciences RPN2232) using a Fusion Fx device (Vilbert Lourmat).

Results

Antibodies were tested on the GST-antigen fusion as a positive control, lysates from wild-type AX2 (Ka) D. discoideum cells, and as negative control a clone of tsg101 knock-out cells (López-Jiménez et al., 2018). Antibodies MRB155, MRB156 and MRB189 recognize the GST-Tsg101 antigen, but not the endogenous Tsg101 in D. discoideum cells (Fig. 1). Antibodies were then tested on lysates from wild-type cells overexpressing GFP-Tsg101. As a control, the J81 antibody that specifically recognizes Tsg101 (Fig. 2) was used. The J81 antibody recognized both the endogenous and exogenous Tsg101, while MRB155, MRB156 and MRB189 did not.

Figure 1. Western blot with MRB155, MRB156, MRB189 on GST-Tsg101, lysates from wild-type AX2(Ka) and tsg101 KO cells.

Figure 2. Western blot with MRB155, MRB156, MRB189 and J81 on lysates from wild-type AX2(Ka), tsg101 KO and AX2(Ka) expressing GFP-Tsg101 cells.

Conflict of interest

The authors declare no conflict of interest.