Anti-POLE (mutated P286R) antibody [EPR28640-56]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

Immunohistochemical analysis of paraffin-embedded (A) 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®. (B) 293T cells transfected with a human wild-type POLE expression vector containing a myc-His-tag®. tissue labeling POLE (mutated P286R) with ab318136 at 1/2000 (0.268 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on (A) 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, no staining on (B) 293T cells transfected with a human wild-type POLE expression vector containing a myc-His-tag®.
The section was incubated with ab318136 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument
Incubate slides with 3% Hydrogen Peroxide for 10 mins at room temperature after secondary antibody incubation to reduce the background

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-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

Flow cytometric analysis of 4% paraformaldehyde fixed 0.1% Tween-20 permeabilized 293T (human embryonic kidney epithelial cell) transfected with a human POLE (mutated P286R) containing a myc-His-tag® (upper right) / 293T transfected with a human wild-type POLE expression vector containing a myc-His-tag® (bottom left) / 293T transfected with an empty vector containing a myc-His-tag® (bottom right). cells labelling POLE (mutated P286R) with ab318136 at 1/500 dilution (0.1ug )/ Upper right, bottom left and bottom right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling POLE (mutated P286R) with ab318136 at 1/100 (5.36 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 293T cells (shown in green) transfected with a human POLE (mutated P286R) expression vector containing a Myc tag, and negative staining in 293T cells transfected with a wildtype POLE expression vector. 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).

ab223894 Anti-Myc tag mouse monoclonal antibody (Alexa Fluor® 594) was used to counterstain tubulin at 1/100 5ug/ml 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 2 ug/mL dilution.

• IP

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Immunoprecipitation - Anti-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

"

POLE (mutated P286R) was immunoprecipitated from 0.35 mg 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, whole cell lysate with ab318136 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318136 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, whole cell lysate
Lane 2 : ab318136 IP in 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318136 in 293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, whole cell lysate

"

Lane 2:

Immunoprecipitation - Anti-POLE (mutated P286R) antibody [EPR28640-56] (ab318136) at 1/30 dilution

Lane 2:

Immunoprecipitation - Anti-POLE (mutated P286R) antibody [EPR28640-56] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/pole-mutated-p286r-antibody-epr28640-56-bsa-and-azide-free-ab318137'>ab318137</a>) at 1/30 dilution

All lanes:

293T cells transfected with a human POLE (mutated P286R) expression vector containing a myc-His-tag®, 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: 67s

• WB

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Western blot - Anti-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-POLE (mutated P286R) antibody [EPR28640-56] (ab318136) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a human wild-type POLE expression vector containi a myc-His-tag®, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a human POLE (mutated P286R) expression vector containi a myc-His-tag®, 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: 261 kDa,124 kDa

false

Exposure time: 180s

• Dot

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Dot Blot - Anti-POLE (mutated P286R) antibody [EPR28640-56] (AB318136)

Dot blot analysis of POLE (mutated P286R) using ab318136 at 1 : 1000 (0.536 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

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

Lanes 1 - 3:

Dot Blot - Anti-POLE (mutated P286R) antibody [EPR28640-56] (ab318136) at 1/1000 dilution

Lanes 1 - 3:

Dot Blot - Anti-POLE (mutated P286R) antibody [EPR28640-56] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/pole-mutated-p286r-antibody-epr28640-56-bsa-and-azide-free-ab318137'>ab318137</a>) at 1/1000 dilution

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