Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 568) (ab175470)

ICC/IF image of ab6046 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA/10% normal donkey serum/0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab6046, 1µg/ml) overnight at +4°C. The secondary antibody (yellow) was ab175470, Alexa Fluor^® 568 Donkey anti-Rabbit IgG - H&L, used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Alexa Fluor^® 568 is pseudocolored in yellow.

HeLa cells showing negative staining by ICC/IF using only secondary antibody. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal donkey serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The secondary antibody (yellow) was ab175470, Alexa Fluor® 568 Donkey anti-Rabbit IgG - H&L, used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

 

Alexa Fluor® 568 is pseudocolored in yellow.

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).