Anti-Cdk4 antibody [EPR4513-32-7]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

ab108357 staining CDK4in the human cell line MCF7 (human breast adenocarcinoma cell line) by intracellular 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)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

Intracellular 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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.

• WB

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Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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.

Lanes 1 - 4:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357)

Lanes 1, 3 and 5:

Wild-type HAP1 cell lysate at 20 µg

Lanes 2, 4 and 6:

CDK4 knockout HAP1 cell lysate at 20 µg

Predicted band size: 34 kDa

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• WB

Lab

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

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.

All lanes:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CDK4 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CDK4 knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cdk4-knockout-hela-cell-line-ab255378'>ab255378</a>)

Predicted band size: 34 kDa,55 kDa

Observed band size: 34 kDa,55 kDa

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• WB

AbReview45217****

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

All lanes:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/1000 dilution

Lane 1:

Human osteosarcoma whole cell lysate - control, non-targeting siRNA at 20 µg

Lane 2:

Human osteosarcoma whole cell lysate - siRNA for CDK4 at 20 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG polyclonal at 1/2000 dilution

Predicted band size: 34 kDa

Observed band size: 34 kDa

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Exposure time: 5s

This image is courtesy of an Abreview submitted by Sonia Rocha

• WB

Lab

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] (ab108357) at 1/10000 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate at 20 µg

Lane 2:

K562 (human chronic myelogenous leukemia cell line from bone marrow) cell lysate at 20 µg

Lane 3:

Ramos (human Burkitt's lymphoma cell line) cell lysate at 20 µg

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

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• OI-RD Scanning

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OI-RD Scanning - Anti-Cdk4 antibody [EPR4513-32-7] (AB108357)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.