Anti-PHIP antibody [EPR27150-82]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PHIP KO HeLa (PHIP knockout human cervical adenocarcinoma epithelial cell, Left) / Parental HeLa (Right) cells labelling PHIP with ab309962 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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Immunohistochemical analysis of paraffin-embedded Human breast carcino tissue labeling PHIP with ab309962 at 1/100 (4.92 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human breast carcinoma.The section was incubated with ab309962 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling PHIP with ab309962 at 1/100 (4.92 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human colon.The section was incubated with ab309962 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Immunohistochemical analysis of paraffin-embedded (A) Wild-type Hela ( tissue labeling PHIP with ab309962 at 1/2000 (0.246 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on (A) Wild-type Hela cell pellet, no staining on (B) PHIP knockout Hela (ab265403) cell pellet.The section was incubated with ab309962 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling PHIP with ab309962 at 1/100 (4.92 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human pancreas.The section was incubated with ab309962 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-PHIP antibody [EPR27150-82] (AB309962)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PHIP KO HeLa (human cervical adenocarcinoma epithelial cell) (ab265403) cells labelling PHIP with ab309962 at 1/100 (4.92 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 parental HeLa cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHIP antibody [EPR27150-82] (AB309962)

Immunohistochemical analysis of paraffin-embedded Human lung adenocarc tissue labeling PHIP with ab309962 at 1/100 (4.92 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human lung adenocarcinoma.The section was incubated with ab309962 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

• IP

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Immunoprecipitation - Anti-PHIP antibody [EPR27150-82] (AB309962)

PHIP was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab309962 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309962 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 2 : ab309962 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309962 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-PHIP antibody [EPR27150-82] (ab309962) at 1/30 dilution

All lanes:

HeLa (human cervical adenocarcinoma epithelial cell) 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: 3min

• WB

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Western blot - Anti-PHIP antibody [EPR27150-82] (AB309962)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Performed under reducing conditions. In Western blot, ab309962 was shown to bind specifically to PHIP. A band was observed at 250 kDa in wild-type Hela cell lysates whereas no signal observed at this size in PHIP knockout cell line ab265403 (knockout cell lysate ab259039). The band beneath the target band (250 kDa) is likely to be degraded target fragment. In Western blot, anti- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 3 minutes

All lanes:

Western blot - Anti-PHIP antibody [EPR27150-82] (ab309962) at 1/1000 dilution

Lane 1:

Wild-type HeLa(human cervical adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

Western blot - Human PHIP knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-phip-knockout-hela-cell-lysate-ab259039'>ab259039</a>) at 40 µg

Lane 3:

K-562 (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 1/20000 dilution

Observed band size: 250 kDa

false

Exposure time: 3min

• WB

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Western blot - Anti-PHIP antibody [EPR27150-82] (AB309962)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The band beneath the target band (250 kDa) is likely to be degraded target fragment. In Western blot, anti-Vinculin antibody (ab129002) loading control staining at 1/10000 dilution. Exposure time : 48 seconds

All lanes:

Western blot - Anti-PHIP antibody [EPR27150-82] (ab309962) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HeLa transfected with siRNA specifically targeti PHIP 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: 250 kDa

false

Exposure time: 48s

• WB

Supplier Data

Western blot - Anti-PHIP antibody [EPR27150-82] (AB309962)

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 : 59 seconds

All lanes:

Western blot - Anti-PHIP antibody [EPR27150-82] (ab309962) at 1/1000 dilution

All lanes:

293T (human embryonic kidney 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: 250 kDa

false

Exposure time: 59s