Well-defined and characterized immunostaining: Primary anti-rabbit IgG antibody (grey) with 2 copies each of a rabbit Fab- and Fc-specific monoclonal Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the primary rabbit IgG antibody.
Higher resolution with anti-rabbit IgG Nano-Secondaries compared to conventional secondary antibodies: Left: Formation of a small, precise complex of Nanobodies (green) & primary antibody (grey). Right: Formation of a large, arbitrary complex of multiple polyclonal secondaries (green) & primary rabbit antibody.
Alpaca anti-rabbit IgG VHH Alexa Fluor? 488 was applied in conjunction with rabbit anti-Lamin B1 antibodies for immunostaining of nuclear lamina (green) in HeLa cells. Scale bar, 20 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
HeLa cells were immunostained with rabbit anti-Lamin B1 antibodies and alpaca anti-rabbit IgG VHH Alexa Fluor? 568 (1:1,000). Confocal and gated STED images were acquired with a Leica TCS SP8 STED 3X microscope, pulsed depletion with a 775 nm laser. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.
Multiple Nano-Secondaries can be applied for multiplex fluorescent Western blotting. This allows multiple targets to be analyzed simultaneously on the same blot at the same time. Multiplex fluorescent Western blot of GFP-TOM70, ?-Tubulin, and GFP in HEK293T cell lysate. Western blot membrane was simultaneously incubated with primary antibodies and Nano-Secondaries. Green: rabbit anti-GFP (ChromoTek PABG1) + alpaca anti-rabbit IgG VHH Alexa Fluor? 488. Magenta: mouse anti-?-Tubulin + alpaca anti-mouse IgG2b VHH Alexa Fluor? 647.
Western blot analysis of EGFP (EGFP-250, ChromoTek) added to HEK293T cell lysate. Detection with rabbit anti-GFP antibody PABG1 (ChromoTek) and alpaca anti-rabbit IgG VHH Alexa Fluor? 488.
Anti-mouse IgG1 Nano-Secondary: Well-defined and characterized immunostaining. Primary anti-mouse IgG1 antibody (grey) with 2X2 monoclonal mouse Fc- specific Nanobodies (green) bound. In total, 8 fluorophores (red stars) label the mouse IgG1 primary antibody.
The anti-mouse IgG1 Nano-Secondary is subclass-specific and does not cross-react with IgGs from other commonly used species (here rabbit) and with mouse IgG2b and IgG3 subclasses.
One-step immunostaining is the simultaneous incubation of mouse IgG1 primary antibody and anti-mouse IgG1 Nano-Secondary. This method reduces incubation and hands-on time. Simultaneous incubation also supports multiplexing, tissue penetration, and cell staining for flow cytometry.
HeLa cells were immunostained with mouse IgG1 anti-αTubulin antibody + alpaca anti-mouse IgG1 VHH Alexa Fluor? 568 (red). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Images were recorded at the Core Facility Bioimaging at the Biomedical Center, LMU Munich.