All lanes : HRP Anti-IKB alpha antibody [E130] (ab202646) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : NFKBIA (IKB alpha) knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 36 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 12 minutes

ab202646 was shown to recognize IKB alpha in wild-type HAP1 cells as signal was lost at the expected MW in NFKBIA (IKB alpha) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NFKBIA (IKB alpha) knockout samples were subjected to SDS-PAGE. Ab202646 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 IKB alpha staining in a section of formalin-fixed paraffin-embedded normal human kidney*, 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 ab202646, 1/50 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

All lanes : HRP Anti-IKB alpha antibody [E130] (ab202646) at 1/5000 dilution

Lane 1 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate
Lane 4 : Liver (Human) Tissue Lysate - fetal normal tissue

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 36 kDa


Exposure time: 4 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 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 ab202646 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.