Anti-Dishevelled 2 antibody [EPR30298-73]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Wildtype A549 (human lung carcinoma epithelial cell) DVL2 KO A549 (DVL2 knockout A549), ab277903 cells labelling Dishevelled 2 with ab326929 at 1/200 (2.615 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing mainly cytoplasmic staining in wildtype A549 cells (shown in green), showing no staining in DVL2 knockout A549 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).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 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 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293 (human embryonic kidney epithelial cell) HL-60 (human acute promyelocytic leukemia promyeloblast) cells labelling Dishevelled 2 with ab326929 at 1/200 (2.615 μg/ml) dilution , followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic condensate staining in HEK-293 cells (shown in green). The counterstain was observed in magenta. The localisation of DVL2 to condensates in HEK293T cells has been described in the literature (PMID : 35867833). Nuclear DNA was labelled with DAPI (shown in blue). Low expression : HL-60. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 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 dilution.

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

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

Low expression : HL-60.

In lanes 3-4, the lysate was stored at -80℃ prior to Western Blotting. The bands beneath the target band (95 kDa) are likey to be degradation products. In lanes 1-2, To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (ab326929) at 1/1000 dilution

Lanes 1 and 3:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lanes 2 and 4:

HL-60 (human acute promyelocytic leukemia promyeloblast) 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 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

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

Performed under reducing conditions.

In Western blot, ab326929 was shown to bind specifically to Dishevelled 2. Target of interest was observed at 95 kDa in wild-type A549 cell lysates (lane 3) with no signal observed at this size in Dishevelled 2 knockout cell line (lane 4) (knockout cell line ab277903 / knockout cell lysate ab288317).

In lane 4, the lysate was stored at -80℃ prior to Western Blotting. The bands beneath the target band (95 kDa) are likey to be degradation products. In lanes 1-3, To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-4 : 59 seconds.

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (ab326929) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Dishevelled 2 knockout A549 whole cell lysate at at 20 µg

Lanes 3 - 4:

HeLa (human cervical adenocarcinoma epithelial cell) 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 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

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

Low expression : liver, lung.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (ab326929) at 1/1000 dilution

Lane 1:

Human ovary tissue lysate at 20 µg

Lane 2:

Human testis tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Lane 4:

Human lung tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 95 kDa,36 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

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

To minimize protein degradation, cells were lysed immediately after harvest and then applied to a gel and transfer membrane for Western blotting as soon as possible.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-73] (ab326929) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HEK-293 transfected with siRNA specifically targeting Dishevelled 2 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 1/20000 dilution

Observed band size: 95 kDa,36 kDa

false

Exposure time: 70s

• Dot

Lab

Dot Blot - Anti-Dishevelled 2 antibody [EPR30298-73] (AB326929)

Dot blot analysis of Dishevelled 2 using ab326929 at 1 : 1000 (0.509 μg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) cells transfected with human Dishevelled 1 expression vector containing a myc-His-tag® whole cell lysate

Lane 2 : 293T cells transfected with human Dishevelled 2 expression vector containing a myc-His-tag® whole cell lysate

Lane 3 : 293T cells transfected with human Dishevelled 3 expression vector containing a myc-His-tag® whole cell lysate

Exposure time : 180 seconds.

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

This antibody does not cross-react with overexpressed human Dishevelled 1 or Dishevelled 3 by dot blot.

All lanes:

Dot Blot - Anti-Dishevelled 2 antibody [EPR30298-73] (ab326929) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) cells transfected with human Dishevelled 1 expression vector containing a myc-His-tag® whole cell lysate

Lane 2:

293T cells transfected with human Dishevelled 2 expression vector containing a myc-His-tag® whole cell lysate

Lane 3:

293T cells transfected with human Dishevelled 3 expression vector containing a myc-His-tag® whole cell lysate

Secondary

All lanes:

Dot 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

false

Exposure time: 180s