ICC/IF image of ab7291 stained HeLa cells. The cells were 4% paraformaldehyde 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 ( ab7291 , 5µg/ml) overnight at +4°C. The secondary antibody (red) was ab150115 Alexa Fluor ^® 647 goat anti-mouse 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 ab8090 (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 goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody ( ab8090 , 0.1μg/1x10 ⁶ cells) for 30 min at 22°C. The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor ^® 647) (ab150115) was used at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] ( ab91361 , 0.1μg/1x10 ⁶ 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.

The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA / 10% normal goat 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 ( ab7291 , 1µg/ml) and ( ab16048 , 1µg/ml) overnight at +4°C. The secondary antibodies were ab150115 Alexa Fluor ^® 647 (red) goat anti-mouse IgG (H+L) used at 2µg/ml for 1h and ab150077 Alexa Fluor ^® 488 (green) goat anti-rabbit IgG (H+L) used at 2µg/ml for 1h. DAPI was used to stain the cell nuclei.

Cross-reactivity of the polyclonal secondary antibody ab182017 was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards at 1 µg/ml (50µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. ab182017 was then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) ( ab6885 ) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT.

Fot the batch tested, ab182017 showed a cross-reactivity below 2% towards Chicken IgY, 6% towards Human IgG, 7% towards Rabbit IgG and 47% towards Rat IgG.

This data was developed using the unconjugated antibody ( ab182017 ).

Cross-reactivity of Goat anti-Mouse IgG H&L ( ab182017 ) and Goat anti-Mouse IgG H&L obtained from two different vendors was tested using a sandwich ELISA approach. The wells were coated with the indicated IgG standards (Rabbit, Human, Mouse and Rat) at 1 µg/ml (50µl/well) and incubated overnight at 4°C, followed by a 5% BSA blocking step for 2h at RT. Secondary antibodies were then added starting at 1 µg/ml and gradually diluted 1/4 (50 µl/well), followed by incubation for 2h. For the detection Donkey anti-Goat IgG H&L (HRP) ( ab6885 ) was used at 1/10,000 dilution (50 µl/well), followed by incubation for 1h at RT. This data is from a representative dilution.

This data was developed using the unconjugated antibody ( ab182017 ).