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

• Flow Cyt

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Flow Cytometry - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (AB150073)

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.

• IHC-Fr

Collaborator

Immunohistochemistry (Frozen sections) - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (AB150073)

ab216324 staining LINE-1 ORF1p in Wt and Maelstrom (Mael) null mutant mouse testis by Immunohistochemistry (Frozen sections). Mael is a repressor of LINE-1 transposable elements, so the expression of LINE-1 ORF1p increases in this KO. Tissue was fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 25°C; Samples were incubated with primary antibody (1/100, 1/500 and 1/1000) for 12 hours at 4°C in 3% Donkey Serum in PBS + 0.05% Triton X. Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150073) (1/1000) was used as the secondary antibody.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - 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).

• ELISA

Lab

ELISA - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (AB150073)

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).

• Alexa Fluor®

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Alexa Fluor® - Donkey Anti-Rabbit IgG H&L (Alexa Fluor® 488) (AB150073)