Anti-GPR89a antibody [EPR29290-504]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-GPR89a antibody [EPR29290-504] (AB325319)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling GPR89a with ab325319 at 1/200 (2.605 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in T-47D cell transfected with scrambled siRNA and the signal is decreased in T-47D cells transfected with siRNA targeting GPR89A. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Anti-KDEL mouse monoclonal antibody was used to counterstain at 1/200 (5 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GPR89a antibody [EPR29290-504] (AB325319)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized T-47D (human ductal breast epithelial tumor epithelial cell) cells labelling GPR89a with ab325319 at 1/200 (2.605 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green).

Confocal image showing cytoplasmic staining in T-47D cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab169276 Anti-GM130 mouse polyclonal antibody - cis-Golgi Marker was used to counterstain cis-Golgi at 1/200 (5 ug/ml) dilution, followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) at 1/1000 (2 ug/ml) dilution (Magenta).

-ve control 1 : ab325319 at a 1/200 dilution followed by ab150120 at a 1/1000 dilution.

-ve control 2 : ab169276 at a 1/200 dilution followed by ab150081 at a 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-GPR89a antibody [EPR29290-504] (AB325319)

GPR89a was immunoprecipitated from 0.35 mg T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate with ab325319 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325319 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-GPR89a antibody [EPR29290-504] (ab325319) at 1/1000 dilution

Lane 1:

(Input) T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate

Lane 2:

ab325319 at 1/30 IP in T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/ko/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab325319 in T-47D whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/ko/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Observed band size: 35 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-GPR89a antibody [EPR29290-504] (AB325319)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregation.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-GPR89a antibody [EPR29290-504] (ab325319) at 1/1000 dilution

Lanes 1 - 2:

T-47D (human ductal breast epithelial tumor epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lanes 1 - 2:

T-47D transfected with siRNA specifically targeting GPR89a whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 35 kDa,36 kDa

false

Exposure time: 62s

• WB

Supplier Data

Western blot - Anti-GPR89a antibody [EPR29290-504] (AB325319)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Samples are non-boiled as boiling may cause protein aggregation.

Lanes 1-4 are incubated with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 and lane 5 is incubated with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

The identity of the bands below 25 kDa in lanes 4-5 are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lanes 1-4 : 5 seconds; Lane 5 : 26 seconds

All lanes:

Western blot - Anti-GPR89a antibody [EPR29290-504] (ab325319) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeting GPR89a whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

T-47D (human ductal breast epithelial tumor epithelial cell) whole cell lysate at 20 µg

Lane 5:

Human cerebellum tissue lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Lane 5:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 35 kDa,36 kDa

false