Anti-Cdk6抗体[8H4] (ab54576)

All lanes : Anti-Cdk6 antibody [8H4] (ab54576) at 1 µg/ml

Lane 1 : Wild-type HCT 116 cell lysate
Lane 2 : CDK6 knockout HCT 116 cell lysate
Lane 3 : Wild-type HeLa ab255929 cell lysate
Lane 4 : CDK6 knockout HeLa ab260923 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 37 kDa

Western blot: Anti-CDK6 antibody [8H4] (ab54576) staining at 1 ug/ml, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab54576 was shown to bind specifically to CDK6. A band was observed at 37 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CDK6 knockout cell line. To generate this image, wild-type and CDK6 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes : Anti-Cdk6 antibody [8H4] (ab54576)

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CDK6 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 37 kDa
Observed band size: 37 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab54576 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) observed at 50 kDa.

 ab54576 was shown to react with Cdk6 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab266059 (knockout cell lysate ab257088) was used. Wild-type HeLa and CDK6 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. ab54576 and Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker (ab52866) overnight at 4°C at a 1 µg/ml and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^®800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^®680RD) preadsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Lanes 1-2 : Anti-Cdk6 antibody [8H4] (ab54576) at 1 µg
Lanes 3-4 : Anti-beta Actin antibody (ab8227) at 1/2000 dilution

Lanes 1 & 3 : Wild-type HAP1 cell lysate
Lanes 2 & 4 : CDK6 knockout HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 37 kDa

Lanes 1 and 2: Green signal from target – ab54576 observed at 37 kDa
Lanes 3 and 4: Red signal from loading control – ab8227 observed at 42 kDa
Lanes 5 and 6: Merged (red and green) signal

ab54576 was shown to react with CDK6 when CDK6 knockout samples were used, along with additional cross-reactive bands. Wild-type and CDK6 knockout samples were subjected to SDS-PAGE. ab54576 and ab8227 (loading control to beta actin) were diluted 1 µg/mL and 1/2000 respectively and incubated overnight at 4ºC. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye^® 680RD)  preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

This image was generated using the ascites version of the product.

Cdk6 antibody (ab54576) at 1ug/lane + Jurkat cell lysate at 25ug/lane.

This image was generated using the ascites version of the product.