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AE391 and AF291 antibodies recognize an HA-tagged recombinant protein by immunofluorescence

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AE391 and AF291 antibodies recognize an HA-tagged recombinant protein by immunofluorescence

LIMA, Wanessa Cristina, COSSON, Pierre

Abstract

AE391 and AF291 antibodies against the HA tag recognize an HA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

LIMA, Wanessa Cristina, COSSON, Pierre. AE391 and AF291 antibodies recognize an

HA-tagged recombinant protein by immunofluorescence. Antibody Reports , 2020, vol. 3, no. 2, p. e133

DOI : 10.24450/journals/abrep.2020.e133

Available at:

http://archive-ouverte.unige.ch/unige:134092

Disclaimer: layout of this document may differ from the published version.

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doi:10.24450/journals/abrep.2020.e133 Antibody Reports, 2020, vol. 3, e133

This work is licensed under a Creative Commons Attribution 4.0 International License.

Geneva University Library Open Access Publications https://oap.unige.ch/journals/abrep | ISSN 2624-8557

AE391 and AF291 antibodies recognize an HA-tagged recombinant protein by immunofluorescence

Wanessa Cristina Lima, Pierre Cosson

Geneva Antibody Facility, Faculty of Medicine, University of Geneva, 1 rue Michel Servet, CH-1211, Geneva, Switzerland

Abstract

AE391 and AF291 antibodies against the HA tag recognize an HA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.

Introduction

The HA tag is a short peptide derived from the influenza virus hemagglutinin protein (Uniprot #P03435), extensively used for detection and purification of tagged proteins with anti-HA antibodies (Green at al., 1982).

Here, we show that the AE391 and AF291 recombinant antibodies detect an HA-tagged human TAC protein by immunofluorescence in HeLa cells.

Materials & Methods

Antibodies: ABCD_AE391 and ABCD_AF291 antibodies (ABCD nomenclature, web.expasy.org/abcd/;

Lima et al., 2020) were produced by the Geneva Antibody Facility (www.unige.ch/medecine/antibodies/) as mini- antibodies with the antigen-binding scFv fused to a mouse IgG2A Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions of the clones 26/9 (for AE391; Churchill et al., 1994) and 12CA5 (for AF291; Arimori et al., 2017) joined by a peptide linker (GGGS)3. HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco

#12338) were transiently transfected with the vector coding for the scFv-Fc. Supernatants (10 and 70 mg/L for AE391 and AF291, respectively) were collected after 4 days.

Antigen: HeLa cells (growing in DMEM GlutaMAXTM, Gibco #31966; supplemented with 8% Fetal Bovine Serum, Gibco #10270) cultured on glass coverslips (Menzel-Gläser, 22x22 mm) and transiently transfected 2 days before the experiment with an HA-tagged TAC protein (Uniprot #P01589), were used to detect the peptide tag. The HA epitope sequence used was YPYDVPDYASLRS and it was present in the C-terminal cytosolic domain of the fusion protein. An antibody detecting the N-terminal extracellular domain of the TAC protein (AJ519, with rabbit IgG Fc; Arsimoles et al., 2020) was used as a positive control. The HA-tagged TAC protein is expected to be mostly present at the cell surface.

Protocol: The whole procedure was carried out at room temperature. Transfected HeLa cells were rinsed once with PBS, 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 3 min, incubated in PBS + 0.2% (w/v) BSA (PBS-BSA) for 30 min, and then with the tested anti-HA antibodies (final concentration 5 mg/L in PBS-BSA) and AJ519 antibody (final concentration 2.5 mg/L in PBS-BSA) for 1 h. After 3 washes (10 min) with PBS-BSA, cells were incubated for 30 min in PBS-BSA with secondary goat anti-mouse IgG conjugated to AlexaFluor-647 and anti-rabbit IgG conjugated to AlexaFluor-488 (1:300, Molecular Probes,

#A21235 and #A11034, respectively). After 3 washes (10 min) with PBS-BSA, cells were incubated during 10 min with DAPI (1:500, Molecular Probes, #D1306), washed twice with PBS-BSA and once with PBS, and 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 63x Neofluar oil immersion objective.

Results

AE391 and AF291 antibodies specifically detected a signal at the plasma membrane in cells transfected with the HA-tagged TAC protein (Fig. 1). The signal co-localized with the signal generated by the anti-TAC AJ519 antibody (Fig. 1, arrows); the specificity of the signal was further verified by the absence of both anti-TAC and anti-HA stainings in the few non-transfected cells (Fig. 1, arrowheads). No staining was observed when the primary antibody was omitted (Fig. 1, No Ab).

References

Arimori T, Kitago Y, Umitsu M, et al. Fv-clasp: An Artificially designed small antibody fragment with improved production compatibility, stability, and crystallizability. Structure 2017; 25(10):1611-1622.

PMID:28919443.

Arsimoles D, D’Esposito A, Gaspoz V, et al. The AJ519 antibody labels the human TAC/IL2RA protein by immunofluorescence. Antibody Reports, 2020, 3:e118.

doi:10.22450/journals/abrep.2020.e118

Churchill ME, Stura EA, Pinilla C, et al. Crystal structure of a peptide complex of anti-influenza peptide antibody Fab 26/9. Comparison of two different antibodies bound to the same peptide antigen. J Mol Biol. 1994; 241(4):534- 56. PMID:7520084

Green N, Alexander H, Olson A, et al. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982;

28(3):477-87. PMID:6176330

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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doi:10.24450/journals/abrep.2020.e133 Antibody Reports, 2020, vol. 3, e133

This work is licensed under a Creative Commons Attribution 4.0 International License.

Geneva University Library Open Access Publications https://oap.unige.ch/journals/abrep | ISSN 2624-8557

Fig. 1. AE391 and AF291 labeled the plasma membrane of HeLa cells expressing the HA-tagged TAC protein (in white); the signal co- localized (arrows) with the signal generated by the anti-TAC AJ519 antibody (in green); in blue, nuclei were stained with DAPI. No labelling was seen when the primary antibody was omitted, or in non-transfected cells (arrowheads). Scale bar: 20 µm.

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