RB252, RB253, RB254 and RB255 antibodies recognize human Miner1 protein by immunofluorescence

Abstract

The recombinant antibodies RB252, RB253, RB254 and RB255 detect by immunofluorescence the human Miner1 protein in paraformaldehyde-fixed cells.

Introduction

Miner1/CISD2 (MitoNEET-related 1 protein, CDGSH iron-sulfur domain-containing protein 2; UniProt #Q8N5K1) is an integral protein of the endoplasmic reticulum (Wiley et al., 2007). Here we describe the ability of four recombinant antibodies to successfully recognize Miner1 by immunofluorescence in MEF cells overexpressing Miner1.

Materials & Methods

Antibodies: RB252, RB253, RB254 and RB255 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 (MRB252, MRB253, MRB254 and MRB255). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco #12338) were transiently transfected with the vectors coding for each scFv-Fc. Supernatants (~50 mg/L) were collected after 5 days.

Antigen: The antibodies were raised against a GST protein fused to the 75 C-terminal residues of Miner1 protein (PKKKQQKDSLINLKIQKENPKVVNEINIEDL CLTKAAYCRCWRSKTFPACDGSHNKHNELTGDNVGPLILKKKEV). MEF cells (growing in DMEM GlutaMAX^TM supplemented with 10% Fetal Bovine Serum) cultured on a glass coverslip (Menzel-Gläser, 22x22 mm) transfected 3 days before the experiment with Miner1 were used to detect the full-length protein.

Protocol: The whole procedure was carried out at room temperature. Transfected MEF cells were fixed with PBS + 4% paraformaldehyde (w/v) (Applichem, #A3013) for 30 min, and blocked with PBS + 40 mM ammonium chloride (NH₄Cl) (Applichem, #A3661) for 5 min. Cells were then permeabilized in PBS + 0.2% saponin (w/v) (Sigma, #S7900) for 5 min, washed once (5 min) with PBS + 0.2% (w/v) BSA (PBS-BSA), and incubated for 30 min with the antibody-containing supernatants (dilution 1:10). After 3 washes (5 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-mouse IgG conjugated to AlexaFluor-488 (1:400, Molecular Probes, #A11029). After 3 washes (5 min) with PBS-BSA, cells were mounted on slides (Menzel-Gläser, 76x26 mm) with Möwiol (Hoechst) + 2.5% (w/v) DABCO (Fluka, #33480). Pictures were taken using a Zeiss LSM700 confocal microscope, with a 40x Neofluar oil immersion objective.

Results

The antibodies MRB252, MRB253, MRB254 and MRB255 specifically detect a signal in Miner1-transfected cells, resembling the endoplasmic reticulum network (Fig. 1). Endogenous Miner1 is detected with a very faint signal. No signal was detected when the primary antibody was omitted (Fig. 1A).

Figure 1. MRB antibodies successfully label MEF cells over-expressing the Miner1 protein. No labelling was seen when the primary antibody was omitted (No Ab panel in A), and a very faint signal (corresponding to endogenous Miner1) can be detected in non-transfected cells. Scale bar: 10 μm.

Conflict of interest

Wanessa Cristina Lima is an editor of the Antibody Reports journal.