All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CDK4 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 34 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab108357 observed at 34 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

 ab108357 was shown to react with Cdk4 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255378 (knockout cell lysate ab263780) was used. Wild-type HeLa and CDK4 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. ab108357 and Anti-Vinculin antibody [VIN-54] 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervix carcinoma tissue labeling Cdk4 with purified ab108357 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer (pH 9). ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L), was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Lanes 1-2 : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution
Lanes 3-4 : Anti-beta Actin antibody [mAbcam 8226] - Loading Control (ab8226) at 1/1000 dilution


Lanes 1 & 3 & 5 : Wild-type HAP1 cell lysate
Lanes 2 & 4 & 6 : CDK4 knockout HAP1 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 34 kDa

Lanes 5 and 6: Merged signal (red and green). Green - ab108357 observed at 34kDa. Red - loading control to beta actin observed at 40kDa.

ab108357 was shown to specifically react with CDK4 in wild-type HAP1 cells. No band was observed when CDK4 knockout samples were examined. Wild-type and CDK4 knockout samples were subjected to SDS-PAGE. ab108357 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 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunocytochemical analysis of MCF7 (human breast adenocarcinoma cell line) cells, labeling Cdk4 with purified ab108357 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500), was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

ab108357 staining CDK4 in the human cell line MCF7 (human breast adenocarcinoma cell line) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/20. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabeled control: Cell without incubation with primary antibody and secondary antibody (Blue)

All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/10000 dilution (purified)

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 2 : K562 (human chronic myelogenous leukemia cell line from bone marrow) cell lysate
Lane 3 : Ramos (human Burkitt's lymphoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 34 kDa
Observed band size: 34 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

ab108357 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 ab108357 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 Cytometry analysis of MCF7 (human breast adenocarcinoma cell line) cells labelling Cdk4 with purified ab108357 at 1/40 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Immunohistochemical analysis of formalin/PFA-fixed paraffin-embedded human urothelial carcinoma tissue labelling Cdk4 with unpurified ab108357 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

All lanes : Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution (unpurified)

Lane 1 : Human osteosarcoma whole cell lysate - control, non-targeting siRNA
Lane 2 : Human osteosarcoma whole cell lysate - siRNA for CDK4

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 34 kDa
Observed band size: 34 kDa


Exposure time: 5 seconds

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