Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 405) (ab175651)

ICC/IF image of ab6046 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes, blocked with Image-iT^® FX Signal Enhancer at room temperature for 30 min and then incubated in 1% BSA / 10% normal donkey serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml) overnight at +4°C. The secondary antibody (blue) was ab175651 Alexa Fluor^® 405 donkey anti-rabbit IgG (H+L) used at 2µg/ml for 1h. DRAQ5 (ab108410) was used to stain the cell nuclei (red) at a concentration of 1.25µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

Cross-reactivity of the polyclonal secondary antibody ab182020 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50 µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182020 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Goat anti-Donkey IgG H&L (HRP) (ab6988) was used at 1/20,000 dilution (50 µl/well), followed by incubation for 1h at RT.

For the batch tested, ab182020 showed a cross-reactivity below 2% towards human IgG, mouse IgG, rat IgG, goat IgG and chicken IgY.

This data was developed using the unconjugated antibody (ab182020).