The RB030 antibody recognizes a peptide of the D. discoideum TspB protein by western blot

Authors

  • Madeleine Zufferey
  • Cedric Blanc

DOI:

https://doi.org/10.24450/journals/abrep.2020.e152

Abstract

The recombinant antibody RB030 detects by western blot a peptide of the Dictyostelium discoideum TspB protein fused to a GST protein; RB029 does not.

Introduction

Tetraspanins, characterized by four transmembrane domains and a large extracellular loop (EC2) containing a conserved CCG-motif, potentially function as regulators of cellular signaling (Albers et al., 2016). Here we describe the ability of the RB030 antibody to detect by western blot a fragment of the TspB protein (DDB_G0269872, UniProt #Q55CW7), one of the five members of the Dictyostelium tetraspanin family, fused to a GST protein.

Materials & Methods

Antibodies: ABCD_RB029 and ABCD_RB030 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 (MRB029 and MRB030). HeLa cells (growing in DMEM GlutaMAXTM (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 47 residues (ENFLKCCYWNSTSSRNPL LCPKDSKGIPKYTDTCDSVISSKISSN) of the TspB EC2 domain. This chimeric GST-TspB protein was used as antigen for detection. GST was used as a negative control.

Protocol: Expression of the GST-TspB 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

The RB030 antibody specifically recognizes the GST-TspB fusion protein (~30 kDa), as well as a probable partial degradation product at ~18 kDa; it does not bind the GST negative control (Fig. 1). RB029 does not recognize GST-TspB (Fig. 1). Note that the antigen used here encompasses a fragment of an extracellular domain of the TspB protein, and it is presumably not properly folded. It may also lack proper post-translational modifications. Further experiments will be necessary to determine if and in which experimental conditions RB030 recognizes the full-length TspB protein.

Figure 1. Specific binding of the RB030 antibody to the GST-TspB protein (predicted molecular mass ~30 kDa).

Conflict of interest

The authors declare no conflict of interest.

References

Blanc C, Zufferey M, Cosson P. Use of in vivo biotinylated GST fusion proteins to select recombinant antibodies. ALTEX. 2014;31(1):37-42. PMID:24100547

Albers T, Maniak M, Beitz E, von Bülow J. The C isoform of Dictyostelium tetraspanins localizes to the contractile vacuole and contributes to resistance against osmotic stress. PLoS One. 2016; 11(9):e0162065. PMID: 27597994

Lima WC, Gasteiger E, Marcatili P, Duek P, Bairoch A, Cosson P. The ABCD database: a repository for chemically defined antibodies. Nucleic Acids Res. 2020; 48(D1):D261-D264. PMID:31410491

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Published

2020-04-06

Section

Article

How to Cite

1.
Zufferey M, Blanc C. The RB030 antibody recognizes a peptide of the D. discoideum TspB protein by western blot. Antib. Rep. [Internet]. 2020 Apr. 6 [cited 2024 Dec. 12];3(3):e152. Available from: https://oap.unige.ch/journals/abrep/article/view/152