Anti-Pelo antibody [EPR26003-259]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Pelo antibody [EPR26003-259] (AB309344)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervical adenocarcinoma epithelial cell) cells labelling Pelo with ab309344 at 1/500 dilution (0.1 ug)/Red (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). A 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-Pelo antibody [EPR26003-259] (AB309344)

Pelo was immunoprecipitated from 0.35 mg HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab309344 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309344 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 : ab309344 IP in HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309344 in HeLa whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 3 seconds Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Pelo antibody [EPR26003-259] (ab309344) 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: 3s

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Pelo antibody [EPR26003-259] (AB309344)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labelling Pelo with ab309344 at 1/500 (0.94 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 mainly cytoplasmic staining in RAW 264.7 cell line.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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Pelo antibody [EPR26003-259] (AB309344)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling Pelo with ab309344 at 1/500 dilution (0.1 ug)/Red (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-Pelo antibody [EPR26003-259] (AB309344)

Pelo was immunoprecipitated from 0.35 mg RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab309344 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309344 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate Lane 2 : ab309344 IP in RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309344 in RAW264.7 whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-Pelo antibody [EPR26003-259] (ab309344) at 1/30 dilution

All lanes:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) 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: 15s

• WB

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Western blot - Anti-Pelo antibody [EPR26003-259] (AB309344)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The band beneath the target band (45 kDa) is likely to be degraded target fragment. Exposure time : 15 seconds

All lanes:

Western blot - Anti-Pelo antibody [EPR26003-259] (ab309344) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse testis tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate at 20 µg

Lane 4:

Rat liver tissue lysate at 20 µg

Lane 5:

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

Lane 6:

HepG2 (human hepatocellar carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 7:

HeLa (human cervix adenocarcinoma 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 1/100000 dilution

Observed band size: 45 kDa

false

Exposure time: 15s

• WB

Supplier Data

Western blot - Anti-Pelo antibody [EPR26003-259] (AB309344)

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

All the lysates were kindly provided by Dr. Jiahuai Han, School of Life Sciences, Xiamen University.

The band beneath the target band (45 kDa) is likely to be degraded target fragment.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-Pelo antibody [EPR26003-259] (ab309344) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

PELO knockout RAW264.7 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: 45 kDa

true

Exposure time: 114s