Anti-POLE antibody [EPR28639-64]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-POLE antibody [EPR28639-64] (AB318968)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized K-562 (human chronic myelogenous leukemia lymphoblast) cells labelling POLE with ab318968 at 1/50 dilution (1 ug)/Magenta 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-POLE antibody [EPR28639-64] (AB318968)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Parental HAP1(human chronic myelogenous leukemia near-haploid cell, Left) / POLE KO HAP1(Right) cells labelling POLE with ab318968 at 1/50 dilution (1ug) / Magenta 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-POLE antibody [EPR28639-64] (AB318968)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized POLE KO HAP 1 (POLE knockout human chronic myelogenous leukemia near-haploid cell) cells labelling POLE with ab318968 at 1/500 (1.07 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 wildtype HAP1 cells and negative staining in POLE knockout HAP1 cells(shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). 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 (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 antibody [EPR28639-64] (AB318968)

POLE was immunoprecipitated from 0.35 mg K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab318968 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318968 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : ab318968 IP in K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318968 in K-562 whole cell lysate

The bands beneath the target band between 100 kDa and 260 kDa are likely to be degraded target fragments.

All lanes:

Immunoprecipitation - Anti-POLE antibody [EPR28639-64] (ab318968) at 1/30 dilution

Lanes 1 - 2:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 10 µg

Lanes 4 - 5:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 10 µg

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: 10s

• IP

Supplier Data

Immunoprecipitation - Anti-POLE antibody [EPR28639-64] (AB318968)

POLE was immunoprecipitated from 0.35 mg parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate with ab318968 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab318968 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate
Lane 2 : ab318968 IP in parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab318968 in parental HAP1(HOWT01) whole cell lysate

The bands beneath the target band between 100 kDa and 260 kDa are likely to be degraded target fragments.

All lanes:

Immunoprecipitation - Anti-POLE antibody [EPR28639-64] (ab318968) at 1/30 dilution

Lanes 1 - 2:

parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 10 µg

Lanes 4 - 5:

POLE Knockout HAP1(HO010404) whole cell lysate at 10 µg

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: 6s

• WB

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Western blot - Anti-POLE antibody [EPR28639-64] (AB318968)

The identity of the band at 100 kDa is unknown.

The bands beneath the target band between 100 kDa and 260 kDa are likely to be degraded target fragments.

In Western blot, ab318968 was shown to bind specifically to POLE. Target of interest was observed at 260kDa in wild-type HAP1 cell lysates (Lane 1) with no signal observed at this size in POLE knockout cell line (Lane 2).

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

All lanes:

Western blot - Anti-POLE antibody [EPR28639-64] (ab318968) at 1/1000 dilution

Lane 1:

parental HAP1(HOWT01) (human chronic myelogenous leukemia near-haploid cell ) whole cell lysate at 20 µg

Lane 2:

POLE Knockout HAP1(HO010404) 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: 260 kDa,124 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-POLE antibody [EPR28639-64] (AB318968)

The identity of the band at 100 kDa is unknown.

The bands beneath the target band between 100 kDa and 260 kDa (Lane 1-8) are likely to be degraded target fragments.

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

Exposure time : Lane 1-8 : 15 seconds; Lane 9-12 : 48 seconds

All lanes:

Western blot - Anti-POLE antibody [EPR28639-64] (ab318968) at 1/1000 dilution

Lane 1:

HeLa (human cervical 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:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

THP-1 (human monocytic leukemia monocyte) whole cell lysate at 20 µg

Lane 5:

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

Lane 6:

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

Lane 7:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 8:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Lane 9:

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

Lane 10:

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

Lane 11:

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

Lane 12:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell fresh 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: 260 kDa,124 kDa

false

• WB

Supplier Data

Western blot - Anti-POLE antibody [EPR28639-64] (AB318968)

The identity of the band at 100 kDa is unknown.

The bands beneath the target band between 100 kDa and 260 kDa (Lane 1-4) are likely to be degraded target fragments.

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

Exposure time : Lane 1-4 : 15 seconds; Lane 4 : 180 seconds

All lanes:

Western blot - Anti-POLE antibody [EPR28639-64] (ab318968) at 1/1000 dilution

Lane 1:

Mouse spleen tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Mouse placenta tissue lysate at 20 µg

Lane 4:

Rat testis tissue lysate at 20 µg

Lane 5:

Mouse testis fresh tissue 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: 260 kDa,124 kDa

false