Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 647) (ab150075)

ICC/IF image of ab6046 stained HeLa cells. The cells were 100% 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 primary antibody (ab6046, 5µg/ml) overnight at +4°C. The secondary antibody (red) was ab150075 Alexa Fluor® 647 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.

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® 647) (ab150075) was used at 1/2000 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 solid-state 25mW red diode laser (635nm) and 675/30 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).

ab62050 staining prostaglandin E synthase in mouse testis tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde, permeabilized with Triton X-100, and blocked with 10% serum for 30 minutes at 24°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/50) for 20 hours at 4°C. An Alexa Fluor® 647-conjugated donkey anti-rabbit IgG H&L (ab150075) (1/200) was used as the secondary antibody.

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