Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073)

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 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 (green) was ab150073 Alexa Fluor^® 488 donkey anti-rabbit IgG (H+L) used at 1µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

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

This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10min).

Overlay histogram showing Jurkat cells stained with ab16669 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal donkey serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab16669, 1/1000 dilution) for 30 min at 22°C. The secondary antibody Donkey anti-rabbit IgG H&L (Alexa Fluor® 488) (ab150073) was used at 1/4000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

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).

ab104603 staining DYNLL1 in mouse cervical spinal cord tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 10% serum for 1 hour at 21°C. Samples were incubated with primary antibody (2 µg/ml in PBS/10% serum/0.1% Triton X-100) for 16 hours at 4°C. An Alexa Fluor^® 488-conjugated Donkey anti-rabbit IgG H&L (ab150073) (1/500) was used as the secondary antibody.