Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / CTNNB1 (beta Catenin) knockout HAP1(Left) cells labelling beta Catenin non-phospho(active) S45 with ab305261 at 1/500 dilution (0.1ug) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labeling beta Catenin non-phospho(active) S45 with ab305261 at 1/500 (0.908 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on human ovarian cancer. The section was incubated with ab305261 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunohistochemical analysis of paraffin-embedded Panel A. Wild-type H tissue labeling beta Catenin non-phospho(active) S45 with ab305261 at 1/2000 (0.227 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on wild-type HAP1 (Panel A); No staining on CTNNB1 (beta Catenin) knockout HAP1 (Panel B).The section was incubated with ab305261 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. 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-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CTNNB1 (beta Catenin) knockout HAP1 cells labelling beta Catenin non-phospho (active) S45 with ab305261 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous staining in parental HAP1 cell line, and no staining in CTNNB1 (beta Catenin) knockout HAP1 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 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 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling beta Catenin non-phospho(active) S45 with ab305261 at 1/500 (0.908 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on human colon. The section was incubated with ab305261 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

beta Catenin non-phospho(active) S45 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg with ab305261 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305261 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 μg Lane 2 : ab305261 IP in HeLa whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305261 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 50 seconds

All lanes:

Immunoprecipitation - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (ab305261) at 1/30 dilution

All lanes:

HeLa (human cervix adenocarcinoma epithelial cell)

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

Observed band size: 95 kDa

false

Exposure time: 50s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling beta Catenin non-phospho(active) S45 with ab305261 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing membranous and weak cytoplasmic staining in NIH/3T3 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 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 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling beta Catenin non-phospho(active) S45 with ab305261 at 1/50 dilution (1ug) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling beta Catenin non-phospho(active) S45 with ab305261 at 1/2000 (0.227 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on rat colon. The section was incubated with ab305261 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. 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-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labeling beta Catenin non-phospho(active) S45 with ab305261 at 1/2000 (0.227 µg/ml) followed by a LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Positive staining on mouse colon. The section was incubated with ab305261 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used at Ready to use dilution. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

beta Catenin non-phospho(active) S45 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 μg with ab305261 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305261 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 μg Lane 2 : ab305261 IP in NIH/3T3 whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305261 in NIH/3T3 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 50 seconds

All lanes:

Immunoprecipitation - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (ab305261) at 1/30 dilution

All lanes:

HeLa (human cervix adenocarcinoma epithelial cell)

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

Observed band size: 95 kDa

false

Exposure time: 50s

• WB

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Western blot - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS The samples were run on a Bis-Tris gel. Performed under reducing conditions. False colour image of Western blot : Anti-beta Catenin non-phospho(active) S45 antibody (ab305261) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody 6C5 (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab305261 was shown to bind specifically to beta Catenin non-phospho(active) S45. A band was observed at 95 kDa in wild-type HAP1 cell lysates with no signal observed at this size in CTNNB1 (beta Catenin) knockout cell line. To generate this image, wild-type and CTNNB1 (beta Catenin) knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (ab305261) at 1/1000 dilution

Lane 1:

Wild-type HAP1(human chronic myelogenous leukemia near-haploid cell line) whole cell lysate at 20 µg

Lane 2:

CTNNB1 (beta Catenin) knockout HAP1 whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Observed band size: 95 kDa

false

• WB

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Western blot - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western Blotting immediately to minimize protein degradation. Exposure time : 180 seconds

All lanes:

Western blot - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (ab305261) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) 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 1/50000 dilution

Observed band size: 95 kDa

false

Exposure time: 180s

• Dot

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Dot Blot - Anti-beta Catenin non-phospho(active) S45 antibody [EPR26155-110-1] (AB305261)

Lane 1 : beta Catenin non-phospho peptide Lane 2 : beta Catenin phospho (S33) peptide Lane 3 : beta Catenin phospho (S33/S37) peptide Lane 4 : beta Catenin phospho (S33/S37/T41) peptide Lane 5 : beta Catenin phospho (S37) peptide Lane 6 : beta Catenin phospho (S37/T41) peptide Lane 7 : beta Catenin phospho (T41) peptide Lane 8 : beta Catenin phospho (S33/T41) peptide Lane 9 : beta Catenin phospho (S33/S37/T41/S45) peptide Lane 10 : beta Catenin phospho (S37/T41/S45) peptide Lane 11 : beta Catenin phospho (T41/S45) peptide Lane 12 : beta Catenin phospho (S45) peptide Lane 13 : beta Catenin phospho (S33/T41/S45) peptide Lane 14 : beta Catenin phospho (S33/S45) peptide Lane 15 : beta Catenin phospho (S37/S45) peptide Dot blot analysis of beta Catenin non-phospho (active) S45 using ab305261 at 1/1000 dilution (0.454 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution. Exposure time : 180 seconds Blocking and diluting buffer and concentration : 5% NFDM/TBST