This data was developed using ab202468, the same antibody clone in a different buffer formulation.

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.

This data was developed using ab202468, the same antibody clone in a different buffer formulation.

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.

This data was developed using ab202468, the same antibody clone in a different buffer formulation.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 Rabbit IgG,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, -20ºC, 30min.

This data was developed using ab202468, the same antibody clone in a different buffer formulation.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 : 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 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

This data was developed using ab202468, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

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

This data was developed using ab202468, the same antibody clone in a different buffer formulation.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.

All lanes : Anti-PDK1 antibody [EPR19571] (ab202468) at 1/2000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Mouse spleen lysate
Lane 4 : Mouse heart lysate
Lane 5 : Rat brain lysate
Lane 6 : Rat kidney lysate
Lane 7 : Rat heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 49 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

This data was developed using ab202468, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: Lanes 1-3, 5, and 6: 8 seconds; Lane 4: 3 seconds; Lane 7: 1 second.