IHC image of HLA A staining in a section of formalin-fixed paraffin-embedded normal human tonsil*, 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 ab199555, 1/100 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-HLA A antibody [EP1395Y] (ab199555) at 1/10000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : HLA-A knockout A431 cell lysate
Lane 3 : A549 cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 41 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab199555 observed at 40 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab199555 was shown to react with HLA A (HRP) in wild-type A431 cells in western blot. Loss of signal was observed when HLA-A knockout sample was used. Wild-type and HLA-A knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab199555 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

HRP Anti-HLA A antibody [EP1395Y] (ab199555) at 1/5000 dilution + Raji (Human Burkitt's lymphoma cell line) Whole Cell Lysate at 10 µg

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 41 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?


Exposure time: 1 minute

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 ab199555 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.