Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (AB236042)

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling Cdk4 with Purified ab68266 at 1 : 100 (5.4 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with None. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 µg/ml) dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (AB236042)

Unpurified ab68266 staining Cdk4 in wild-type HAP1 cells (top panel) and Cdk4 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 ab68266 at 1/250 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (AB236042)

Intracellular Flow Cytometry analysis of A549 (Human lung carcinoma epithelial cell) cells labeling Cdk4 with purified ab68266 at 1/50 dilution (1 μg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

• IP

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Immunoprecipitation - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (AB236042)

ab68266 (purified) at 1 : 30 dilution (2μg) immunoprecipitating Cdk4 in K562 whole cell lysate.
Lane 1 (input) : K562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10μg
Lane 2 (+) : ab68266 & K562 whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab68266 in K562 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

All lanes:

Immunoprecipitation - Anti-Cdk4 antibody [EPR2513Y] (<a href='/en-us/products/primary-antibodies/cdk4-antibody-epr2513y-ab68266'>ab68266</a>)

Predicted band size: 34 kDa

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• WB

Supplier Data

Western blot - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (AB236042)

Lanes 1, 3 and 5 : Wild-type HAP1 cell lysate (20 μg)
Lanes 2, 4 and 6 : CDK4 knockout HAP1 cell lysate (20 μg)
Lanes 1 and 2 : Green signal from target - ab68266 observed at 34 kDa
Lanes 3 and 4 : Red signal from loading control - ab8226 observed at 42 kDa
Lanes 5 and 6 : Merged (red and green) signal
Unpurified ab68266 was shown to specifically react with CDK4 when CDK4 knockout samples were used. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab68266 and ab8226 (loading control to beta actin) were both diluted at 1/1000 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 1/10000 dilution for 1 h at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab68266).

All lanes:

Western blot - Anti-Cdk4 antibody [EPR2513Y] - BSA and Azide free (ab236042)

Predicted band size: 34 kDa

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