Anti-Proteasome subunit beta type 2/PSMB2 antibody [MCP165] (ab22650) at 1/1000 dilution + HeLa cell lysate

Predicted band size: 23 kDa

Lane 1 : Protein Marker IV
Lanes 2-7 : Anti-Proteasome subunit beta type 2/PSMB2 antibody [MCP165] (ab22650) at 1/1000 dilution

Lane 1 : Protein Marker IV
Lanes 2 & 4 & 6 : Protein extract from ciliary body of diseased human donor at 2 µg
Lanes 3 & 5 & 7 : Protein extract from ciliary body of normal human donor at 2 µg

Secondary
Lanes 2-7 : HRP conjugated goat anti mouse antibody

Predicted band size: 23 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?

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IHC image of ab22650 staining in human normal lymphoid formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab22650, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

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.

Overlay histogram showing HeLa cells stained with ab22650 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab22650, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.