Anti-PDK1 antibody [EPR19571]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PDK1 antibody [EPR19571] (AB202468)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PDK1 with ab202468 at 1/1000 dilution followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab202468 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PDK1 antibody [EPR19571] (AB202468)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) cells labeling PDK1 with ab202468 at 1/1000 dilution followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on RAW 264.7 cell line.

The nuclear counterstain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows : -

-ve control 1 : ab202468 at 1/1000 dilution followed by ab150120 at 1/1000 dilution.

-ve control 2 : ab7291 at 1/1000 dilution followed by ab150077 at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-PDK1 antibody [EPR19571] (AB202468)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PDK1 with ab202468 at 1/600 dilution (red) compared with a RabbitIgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.

Cells were permeabilised by 90% methanol-1XPBS, -20°C, 30min.

• IP

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Immunoprecipitation - Anti-PDK1 antibody [EPR19571] (AB202468)

PDK1 was immunoprecipitated from 1mg of NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate with ab202468 at 1/40 dilution.

Western blot was performed from the immunoprecipitate using ab202468 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : NIH/3T3 whole cell lysate, 10μg (Input).

Lane 2 : ab202468 IP in NIH/3T3 whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal -[EPR25A] - Isotype Control (ab172730) instead of ab202468 in NIH/3T3 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-PDK1 antibody [EPR19571] (ab202468)

Predicted band size: 49 kDa

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• WB

Lab

Western blot - Anti-PDK1 antibody [EPR19571] (AB202468)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : PDK1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : Jurkat whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab202468 observed at 45 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab202468 was shown to specifically react with PDK1 in wild-type cells along with additional cross reactive bands. Signal was lost in PDK1 knockout cells. Wild-type and PDK1 knockout samples were subjected to SDS-PAGE. ab202468 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19571] (ab202468)

Predicted band size: 49 kDa

false

• WB

Supplier Data

Western blot - Anti-PDK1 antibody [EPR19571] (AB202468)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1 : 1 minute; Lane 3-4 : 10 seconds; Lane 5/6 : 8 seconds.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19571] (ab202468) at 1/2000 dilution

Lane 1:

L-929 (Mouse connective tissue fibroblast cell line) whole cell lysate

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lane 3:

F9 (Mouse embryonic testicular cancer cell line) whole cell lysate

Lane 4:

bEnd.3 (Mouse brain endothelioma cell line) whole cell lysate

Lane 5:

C6 (Rat glial tumor cell line) whole cell lysate

Lane 6:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate

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

Predicted band size: 49 kDa

Observed band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-PDK1 antibody [EPR19571] (AB202468)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2/3/5/6 : 8 seconds; Lane 4 : 3 seconds; Lane 7 : 1 second.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19571] (ab202468) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Mouse heart lysate at 10 µg

Lane 5:

Rat brain lysate at 10 µg

Lane 6:

Rat kidney lysate at 10 µg

Lane 7:

Rat heart lysate at 10 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

Predicted band size: 49 kDa

Observed band size: 47 kDa

false