Anti-Cdk6 抗体 [EPR4515]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk6 antibody [EPR4515] (AB124821)

ab124821 staining Cdk6 in wild-type HAP1 cells (top panel) and Cdk6 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab124821 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cdk6 antibody [EPR4515] (AB124821)

Overlay histogram showing HeLa cells stained with ab124821 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab124821, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cdk6 antibody [EPR4515] (AB124821)

ICC/IF image of ab124821 at 1/100 dilution, staining Cdk6 in HeLa cells.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk6 antibody [EPR4515] (AB124821)

ab124821 at 1/100 dilution, staining Cdk6 in Formalin fixed Paraffin-embedded Human tonsil tissue by Immunohistochemistry.

Heat mediated antigen retrieval was performed with 0.01M citrate buffer, pH 6.0.

• WB

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Western blot - Anti-Cdk6 antibody [EPR4515] (AB124821)

All lanes:

Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

K562 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

293T cell lysate at 10 µg

Secondary

All lanes:

Goat-anti-rabbit HRP at 1/2000 dilution

Predicted band size: 37 kDa

Observed band size: 37 kDa

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• WB

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Western blot - Anti-Cdk6 antibody [EPR4515] (AB124821)

Lanes 1 and 2 : Green signal from target - ab124821 observed at 37 kDa
Lanes 3 and 4 : Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal
ab124821 was shown to specifically react with CDK6 when CDK6 knockout samples were used. Wild-type and CDK6 knockout samples were subjected to SDS-PAGE. ab124821 and ab8226 (loading control to beta actin) were diluted at 1/10 000 and 1/1000 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 h at room temperature before imaging.

Lanes 1 - 2:

Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821) at 1/10000 dilution

Lanes 3 - 4:

Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821)

Lanes 1, 3 and 5:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2, 4 and 6:

CDK6 knockout HAP1 cell lysate at 20 µg

Predicted band size: 37 kDa

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• WB

Lab

Western blot - Anti-Cdk6 antibody [EPR4515] (AB124821)

Lanes 1- 2 : Merged signal (red and green). Green - ab124821 observed at 37 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab124821 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. ab124821 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 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-Cdk6 antibody [EPR4515] (ab124821) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CDK6 knockout HeLa cell lysate at 20 µg

Predicted band size: 37 kDa

Observed band size: 37 kDa

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• WB

Lab

Western blot - Anti-Cdk6 antibody [EPR4515] (AB124821)

Western blot : Anti-CDK6 antibody [EPR4515] (ab124821) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab124821 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-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Cdk6 antibody [EPR4515] (ab124821) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

CDK6 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HeLa ab255929 cell lysate at 20 µg

Lane 4:

CDK6 knockout HeLa ab260923 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 37 kDa

Observed band size: 37 kDa

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