Abstract
AE391 and AF291 antibodies against the HA tag recognize an HA-tagged human TAC protein by immunofluorescence in paraformaldehyde-fixed HeLa cells.
Highlights
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)
Antibodies: ABCD_AE391 and ABCD_AF291 antibodies (ABCD nomenclature, web.expasy.org/abcd/; Lima et al, 2020) were produced by the Geneva Antibody Facility as miniantibodies with the antigen-binding scFv fused to a mouse IgG2A Fc
After 3 washes (10 min) with PBS + 0.2% (w/v) BSA (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)
Summary
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). 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 miniantibodies 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 FreeStyleTM 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.
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