Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

Flow Cytometry analysis of MCF7 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

ab108357 staining CDK4 in the human cell line MCF-7 (human breast carcinoma) 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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

Immunocytochemical analysis of MCF7 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab108357).

• WB

Lab

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

This data was developed using the same antibody clone in a different buffer formulation (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] - BSA and Azide free (ab213216) at 1/1000 dilution

Lanes 5 - 6:

Western blot - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (ab213216)

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] - BSA and Azide free (AB213216)

This data was developed using the same antibody clone in a different buffer formulation (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] (<a href='/en-us/products/primary-antibodies/cdk4-antibody-epr4513-32-7-ab108357'>ab108357</a>) 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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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-Cdk4 antibody [EPR4513-32-7] - BSA and Azide free (AB213216)

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.