Anti-AC3 antibody [EPR28777-12]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-AC3 antibody [EPR28777-12] (AB323453)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized ADCY3 KO HeLa (ADCY3 knockout human cervical adenocarcinoma epithelial cell), ab264709 cells labelling AC3 with ab323453 at 1/2000 (0.263 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 cytoplasmic staining in wildtype HeLa cells (shown in green), showing no staining in ADCY3 knockout HeLa cells. 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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) 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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-AC3 antibody [EPR28777-12] (AB323453)

Immunofluorescent analysis of 80% Methanol-fixed, 0.1% TritonX-100 permeabilized EL4 (mouse lymphoma T lymphocyte) cells labelling AC3 with ab323453 at 1/50 (10.5 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 cytoplasmic staining in EL4 cells (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).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) 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.

• WB

Supplier Data

Western blot - Anti-AC3 antibody [EPR28777-12] (AB323453)

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

The samples are unboiled.

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

All lanes:

Western blot - Anti-AC3 antibody [EPR28777-12] (ab323453) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 10 µg

Lane 2:

HeLa transfected with siRNA specifically targeting ADCY3 whole cell lysate at 10 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-AC3 antibody [EPR28777-12] (AB323453)

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

The samples are unboiled.

In Western blot, ab323453 was shown to bind specifically to ADCY3.

Target of interest was observed at 150 kDa in wild-type HeLa cell lysates (lane 1) with no signal observed at this size in ADCY3 knockout cell line (lane 2).

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

All lanes:

Western blot - Anti-AC3 antibody [EPR28777-12] (ab323453) at 1/1000 dilution

Lane 1:

Wide-type HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

ADCY3 knockout HeLa whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-AC3 antibody [EPR28777-12] (AB323453)

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

The samples are unboiled.

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

All lanes:

Western blot - Anti-AC3 antibody [EPR28777-12] (ab323453) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) whole cell lysate at 40 µg

Lane 2:

786-O (human kidney epithelial cell) whole cell lysate at 40 µg

Lane 3:

Human cerebellum tissue lysate at 40 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-AC3 antibody [EPR28777-12] (AB323453)

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

Low expression : heart(PMID : 9564875), A20

The samples are unboiled.

The identity of the band higher than 150 kDa is unknown.

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

All lanes:

Western blot - Anti-AC3 antibody [EPR28777-12] (ab323453) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 40 µg

Lane 2:

Mouse heart tissue lysate at 40 µg

Lane 3:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 40 µg

Lane 4:

A20 (mouse reticulum sarcoma B lymphocyte) whole cell lysate at 40 µg

Secondary

All lanes:

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

Observed band size: 150 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-AC3 antibody [EPR28777-12] (AB323453)

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

The samples are unboiled.

This antibody does not cross-react with human ADCY1, ADCY2, ADCY4, ADCY5, ADCY6, ADCY7, ADCY8 or ADCY9.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-AC3 antibody [EPR28777-12] (ab323453) at 1/1000 dilution

Lane 1:

293T cells transfected with a human ADCY3 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 2:

293T cells transfected with a human ADCY1 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 3:

293T cells transfected with a human ADCY2 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 4:

293T cells transfected with a human ADCY4 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 5:

293T cells transfected with a human ADCY5 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 6:

293T cells transfected with a human ADCY6 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 7:

293T cells transfected with a human ADCY7 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 8:

293T cells transfected with a human ADCY8 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Lane 9:

293T cells transfected with a human ADCY9 expression vector containing a myc-his-tag, whole cell lysate at 10 µg

Secondary

All lanes:

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

Observed band size: 150 kDa

false

Exposure time: 3s