Anti-ASH2L antibody [EPR28054-48]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ASH2L antibody [EPR28054-48] (AB314128)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling ASH2L with ab314128 at 1/500 (1.01 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human testis. The section was incubated with ab314128 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

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ASH2L antibody [EPR28054-48] (AB314128)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling ASH2L with ab314128 at 1/1000 (0.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on mouse testis. The section was incubated with ab314128 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-ASH2L antibody [EPR28054-48] (AB314128)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized ES-D3 [D3] (mouse blastocyst-derived embryonic stem cell) cells labelling ASH2L with ab314128 at 1/50 (10.1 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 nuclear staining in ES-D3 [D3] cell line. 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 (Red). 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 2 ug/ml dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ASH2L antibody [EPR28054-48] (AB314128)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling ASH2L with ab314128 at 1/1000 (0.505 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on rat testis. The section was incubated with ab314128 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-ASH2L antibody [EPR28054-48] (AB314128)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized ES-D3 (mouse blastocyst-derived embryonic stem cell) cells labelling ASH2L with ab314128 at 1/500 dilution (0.1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-ASH2L antibody [EPR28054-48] (AB314128)

ASH2L was immunoprecipitated from 0.35 mg ES-D3 (mouse embryonic pluripotent stem Cell) whole cell lysate with ab314128 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab314128 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

Lane 1:

ES-D3 (mouse embryonic pluripotent stem Cell) whole cell lysate

Lane 2:

ab314128 IP in ES-D3 (mouse embryonic pluripotent stem Cell) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab314128 in ES-D3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-ASH2L antibody [EPR28054-48] (AB314128)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 15199122).

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

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

All lanes:

Western blot - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

All lanes:

K562 (human chronic myelogenous leukemia lymphoblast) 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: 65 kDa,80 kDa,36 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-ASH2L antibody [EPR28054-48] (AB314128)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 15199122).

Lanes 1-6 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 and lane 7 is applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

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

Exposure time : Lanes 1-6 : 180 seconds, lane 7 : 37 seconds

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

All lanes:

Western blot - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Mouse lung tissue lysate at 20 µg

Lane 4:

Mouse liver tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 µg

Lane 6:

Rat testis tissue lysate at 20 µg

Lane 7:

Human testis tissue lysate at 20 µg

Secondary

Lanes 1 - 6:

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

Lane 7:

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

Observed band size: 65 kDa,80 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-ASH2L antibody [EPR28054-48] (AB314128)

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

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

All lanes:

Western blot - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

Lane 1:

F9 (mouse embryonal carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

F9 transfected with siRNA specifically targeting ASH2L 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: 65 kDa,80 kDa,36 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-ASH2L antibody [EPR28054-48] (AB314128)

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

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

All lanes:

Western blot - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

NIH/3T3 transfected with siRNA specifically targeting ASH2L 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: 80 kDa,36 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-ASH2L antibody [EPR28054-48] (AB314128)

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 15199122).

In lanes 1-3, the lysates were stored at -80℃ prior to Western Blotting. The bands beneath the target band (40 kDa) are likely to be degradation products. In lane 4, the lysate was freshly made and used for Western Blotting immediately to minimize protein degradation.

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

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

All lanes:

Western blot - Anti-ASH2L antibody [EPR28054-48] (ab314128) at 1/1000 dilution

Lanes 1 and 4:

ES-D3 (mouse embryonic pluripotent stem Cell) whole cell lysate at 20 µg

Lane 2:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

MEF (mouse embryonic fibroblast (immortalized)) 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: 65 kDa,80 kDa,36 kDa

false

Exposure time: 37s