Goat Anti-Mouse IgG H&L (Alexa Fluor® 647)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized IMR-90 (human lung fibroblast) cells labelling IDUA with ab323701 at 1/50 (10.2 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing lysosome staining in IMR-90 cells(shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue).
Low expression : JAR.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab25630 Anti-LAMP1 mouse monoclonal antibody was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

Counter stain secondary antibody only control : Secondary antibody is ab150115 Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed at 1/1000 2ug/ml dilution.

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Lab

Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

Immunocytochemistry/Immunofluorescence analysis of A549 (human lung carcinoma epithelial) cells with ab108257 at a 1/100 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at a 1/1000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Mouse anti-COX IV monoclonal antibody - Mitochondrial Marker was used as a counterstain at a 1/50 dilution followed by ab150115 Alexa Fluor^® 647 Goat anti-Mouse secondary antibody used at a 1/1000 dilution.

The negative controls are shown in bottom middle and right hand panels - for negative control 1, ab108257 was used at a dilution of 1/100 followed by Alexa Fluor^® 647 Goat anti-Mouse secondary ab150115 at a dilution of 1/1000. For negative control 2, Mouse anti-COX IV monoclonal antibody - Mitochondrial Marker was used at a dilution of 1/50 followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed ab150081 at a dilution of 1/1000.

Confocal image showing cytoplasmic staining in A549 cells (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

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.

• Flow Cyt

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Flow Cytometry - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

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/1x10⁶ 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/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.

• sELISA

Lab

Sandwich ELISA - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

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.

For 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 were developed using the unconjugated antibody (ab182017).

• sELISA

Lab

Sandwich ELISA - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)

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 were developed using the unconjugated antibody (ab182017).

• Alexa Fluor®

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Alexa Fluor® - Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) (AB150115)