Anti-Cdk8 antibody [EPR21005]

• IP

Unknown

Immunoprecipitation - Anti-Cdk8 antibody [EPR21005] (AB229192)

Cdk8 was immunoprecipitated from 0.35 mg of SW480 (human colorectal adenocarcinoma cell line) whole cell lysate with ab229192 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab229192 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/10,000 dilution.

Lane 1 : SW480 whole cell lysate 10 μg (Input).
Lane 2 : ab229192 IP in SW480 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab229192 in SW480 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-Cdk8 antibody [EPR21005] (ab229192)

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

• WB

Lab

Western blot - Anti-Cdk8 antibody [EPR21005] (AB229192)

Lanes 1-4 : Merged signal (red and green). Green - ab229192 observed at 53 kDa. Red - loading control ab8245 observed at 37 kDa.

ab229192 Anti-Cdk8 antibody [EPR21005] was shown to specifically react with Cdk8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265087 (knockout cell lysate ab257885) was used. Wild-type and Cdk8 knockout samples were subjected to SDS-PAGE. ab229192 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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-Cdk8 antibody [EPR21005] (ab229192) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CDK8 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CDK8 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cdk8-knockout-hela-cell-line-ab265087'>ab265087</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

• WB

Supplier Data

Western blot - Anti-Cdk8 antibody [EPR21005] (AB229192)

Blocking and dilution buffer : 5% NFDM/TBST.

ab229192 was shown to specifically react with Cdk8 in wild-type HAP1 cells as signal was lost in Cdk8 knockout cells.

Wild-type and Cdk8 knockout samples were subjected to SDS-PAGE. ab229192 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

All lanes:

Western blot - Anti-Cdk8 antibody [EPR21005] (ab229192) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Cdk8 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HCT 116 (human colorectal carcinoma cell line) whole cell lysate at 10 µg

Lane 4:

SW480 (Human colorectal adenocarcinoma 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: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 48s

• WB

Supplier Data

Western blot - Anti-Cdk8 antibody [EPR21005] (AB229192)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cdk8 antibody [EPR21005] (ab229192) at 1/1000 dilution

Lane 1:

Human testis lysate at 10 µg

Lane 2:

K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate at 10 µg

Secondary

Lane 1:

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 1/20000 dilution

Lane 2:

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 1/50000 dilution

Predicted band size: 53 kDa

Observed band size: 53 kDa

false

Exposure time: 58s