All lanes : HRP Anti-p27 KIP 1 antibody [Y236] (ab194235) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : CDKN1B (p27 KIP 1) knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 22 kDa


Exposure time: 2 minutes

ab194235 was shown to recognize p27 KIP 1 in wild-type HAP1 cells as signal was lost at the expected MW in CDKN1B (p27 KIP 1) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and CDKN1B (p27 KIP 1) knockout samples were subjected to SDS-PAGE. Ab194235 and ab184095 (Mouse monoclonal [mAbcam 9484] to GAPDH - Loading Control (Alexa Fluor^® 680) loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/1000 dilution respectively. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

IHC image of p27 KIP 1 staining in a section of formalin-fixed paraffin-embedded human colon adenocarcinoma*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab194235 at 1/70 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then 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 non-automated) 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

IHC image of p27 KIP 1 staining in a section of formalin-fixed paraffin-embedded human colon 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 ab194235 at 1µg/ml. 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