Anti-PDK1 antibody [EPR19573]

• IP

Supplier Data

Immunoprecipitation - Anti-PDK1 antibody [EPR19573] (AB207450)

PDK1 was immunoprecipitated from 1mg of HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate with ab207450 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab207450 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HepG2 whole cell lysate 10μg (Input).

Lane 2 : ab207450 IP in HepG2 whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab207450 in HepG2 whole cell lysate.

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

Exposure time : 1 second.

All lanes:

Immunoprecipitation - Anti-PDK1 antibody [EPR19573] (ab207450)

Predicted band size: 49 kDa

false

• WB

Supplier Data

Western blot - Anti-PDK1 antibody [EPR19573] (AB207450)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1-5 : 15 seconds; Lane 6 : 8 seconds.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19573] (ab207450) at 1/2000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 10 µg

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg

Lane 5:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 6:

LNCaP (Human prostate cancer cell line) whole cell 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

• WB

Supplier Data

Western blot - Anti-PDK1 antibody [EPR19573] (AB207450)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19573] (ab207450) at 1/5000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) mitochondria fraction lysate at 10 µg

Lane 2:

HEK-293 (Human epithelial cell line from embryonic kidney) cytosol fraction 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

Exposure time: 3min

• WB

Lab

Western blot - Anti-PDK1 antibody [EPR19573] (AB207450)

Lanes 1- 2 : Merged signal (red and green). Green - ab207450 observed at 45 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab207450 was shown to react with PDK1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264791 (knockout cell lysate ab257164) was used. Wild-type HeLa and PDK1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab207450 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19573] (ab207450) at 1/2000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PDK1 knockout HeLa cell lysate at 20 µg

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

• WB

Lab

Western blot - Anti-PDK1 antibody [EPR19573] (AB207450)

Lanes 1 - 4 : Merged signal (red and green). Green - ab207450 observed at 45 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab207450 was shown to specifically react with PDK1 in wild-type HAP1 cells. No band was observed when PDK1 knockout samples were examined. Wild-type and PDK1 knockout samples were subjected to SDS-PAGE. ab207450 and ab8245 (loading control to GAPDH) were diluted at 1/2000 and 1/10,000 dilution respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19573] (ab207450) at 1/2000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

PDK1 knockout HAP1 cell lysate at 20 µg

Lane 3:

HepG2 cell lysate at 20 µg

Lane 4:

LnCaP cell lysate at 20 µg

Predicted band size: 49 kDa

false

• WB

Unknown

Western blot - Anti-PDK1 antibody [EPR19573] (AB207450)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/3/4 : 8 seconds; Lane 2 : 30 seconds; Lane 5/6 : 3 minutes; Lane 7/8 : 15 seconds.

All lanes:

Western blot - Anti-PDK1 antibody [EPR19573] (ab207450) at 1/2000 dilution

Lane 1:

Human heart lysate at 10 µg

Lane 2:

Human kidney lysate at 10 µg

Lane 3:

Mouse brain lysate at 10 µg

Lane 4:

Mouse heart lysate at 10 µg

Lane 5:

Mouse kidney lysate at 10 µg

Lane 6:

Mouse spleen lysate at 10 µg

Lane 7:

Rat kidney lysate at 10 µg

Lane 8:

Rat spleen lysate at 10 µg

Secondary

Lanes 1 - 2:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Lanes 3 - 8:

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