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

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 Nterminus of Tsg101 were not able to detect the full-length Tsg101 protein by western blot.

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 5x10 7 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.