All lanes : HRP Anti-Cdk4 antibody [EPR4513-32-7] - Loading Control (ab193968) at 1/5000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : CDK4 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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


Exposure time: 30 seconds

ab193968 was shown to react with Cdk4 in wild-type HAP1 cells in Western blot. Loss of signal was observed when CDK4 knockout sample was used. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab193968 overnight at 4°C at a 1 in 5000 dilution and ab184095 (Mouse Anti-GAPDH antibody [mAbcam 9484] - Alexa Fluor^® 680) at a 1 in 1000 dilution. Blots were developed with Optiblot ECL reagent (ab133456) and imaged.

All lanes : HRP Anti-Cdk4 antibody [EPR4513-32-7] - Loading Control (ab193968) at 1/5000 dilution

Lane 1 : HeLa whole cell lysate (ab150035)
Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 3 : K562 (Human erythromyeloblastoid leukemia cell line) Whole Cell Lysate
Lane 4 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

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


Exposure time: 90 seconds

This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab193968 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.

IHC image of Cdk4 staining in a section of formalin-fixed paraffin-embedded human breast adenocarcinoma*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins, and incubated overnight at +4°C with ab193968 at a working dilution of 1 in 500. DAB was used as the chromogen (ab103723), diluted 1/100 and incubated for 10min at room temperature. The section was counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and manual) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.