Anti-TNPO3 antibody [3152C2a] (ab54353) at 1/100 dilution + HeLa whole cell lysate at 25 µg

Secondary
anti Mouse IgG at 1/2500 dilution

Predicted band size: 110 kDa
Observed band size: ~90 kDa why is the actual band size different from the predicted?

Anti-TNPO3 antibody [3152C2a] (ab54353) at 1/50 dilution + NIH3T3 whole cell lysate at 25 µg

Secondary
anti Mouse IgG at 1/2500 dilution

Predicted band size: 110 kDa
Observed band size: ~90 kDa why is the actual band size different from the predicted?

IHC image of ab54353 staining in human normal cervix 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 ab54353, 5µg/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 ab54353 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab54353, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 80% methanol/permeabilized in 0.1% PBS-Tween used under the same conditions.