Anti-Dishevelled 2 antibody [EPR30298-74]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Immunohistochemical analysis of paraffin-embedded Human ovary tissue labeling Dishevelled 2 with ab325939 at 1/1000 (0.509 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Positive staining on human ovary. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND®RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (BOND® Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Immunohistochemical analysis of paraffin-embedded Human cerebrum tissue labeling Dishevelled 2 with ab325939 at 1/1000 (0.509 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Low expression tissue : no staining on human cerebrum (PMID : 21990322). The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling Dishevelled 2 with ab325939 at 1/1000 (0.509 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Positive staining on human ovarian cancer. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Dishevelled 2 with ab325939 at 1/1000 (0.509 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression tissue : no staining on human liver. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type A549 (human lung carcinoma epithelial cell) cell pellet and (B) DVL2 knockout A549 cell pellet labeling Dishevelled 2 with ab325939 at 1/2000 (0.255 µg/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Positive staining on (A) Wild-type A549 cell pellet, no staining on (B) DVL2 knockout A549 cell pellet. The primary antibody was incubated for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND®RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

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

Performed under reducing conditions.

In Western blot ab325939 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).

Low expression : liver lung.

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

Exposure time : Lanes 1-4 : 26 seconds; Lane 5 : 6 seconds; Lane 6 : 26 seconds.

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (ab325939) 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

Lane 5:

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

Lane 6:

Dishevelled 2 knockout A549 whole cell lysate at 20 µg

Lane 7:

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

Lane 8:

NCI-H1299 (human lung carcinoma 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 [EPR30298-74] (AB325939)

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

In lanes 1 and 4, the lysates were stored at -80degC prior to Western Blotting. The bands beneath the target band (95 kDa) are likely to be degradation products. In lanes 2-3, 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).

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (ab325939) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) fresh whole cell lysate at 20 µg

Lane 4:

NIH/3T3 (mouse embryonic fibroblast) frozen 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: 59s

• WB

Lab

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

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

Low expression : brain, liver, spleen.

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

Exposure time : Lanes 1-2 : 180 seconds; Lanes 3-5 : 6 seconds.

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (ab325939) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse liver tissue lysate at 20 µg

Lane 3:

Mouse testis tissue lysate at 20 µg

Lane 4:

Mouse spleen tissue lysate at 20 µg

Lane 5:

L Cells (mouse Subcutaneous connective tissue fibroblast) whole cell lysate at 20 µg

Lane 6:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 7:

CT26.WT (mouse colon fibroblast) 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 [EPR30298-74] (AB325939)

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

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

All lanes:

Western blot - Anti-Dishevelled 2 antibody [EPR30298-74] (ab325939) 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: 59s

• Dot

Lab

Dot Blot - Anti-Dishevelled 2 antibody [EPR30298-74] (AB325939)

Dot blot analysis of Dishevelled 2 using ab325939 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 : 3 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 [EPR30298-74] (ab325939) 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: 3s