IHC image of Islet 1 staining in Human Pancreas formalin fixed paraffin embedded tissue section, performed on a Leica Bond 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 ab86501, 1/1000 dilution, 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.

All lanes : Anti-Islet 1 antibody [1B1] (ab86501) at 1/500 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : Islet 1 knockout HAP1 whole cell lysate
Lane 3 : SH-SY5Y whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 39 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 - ab86501 observed at 40 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab86501 was shown to recognize Islet 1 in wild-type HAP1 cells as signal was lost at the expected MW in Islet 1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and Islet 1 knockout samples were subjected to SDS-PAGE. ab86501 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Confocal immunofluorescence analysis of HEK293 cells trasfected with full-length Islet 1-hIgGFc using ab86501 at 1/150 dilution (green). Blue: DRAQ5 fluorescent DNA dye.

Anti-Islet 1 antibody [1B1] (ab86501) at 1/500 dilution + full length Islet 1-hIgGFc transfected HEK293 cell lysate

Predicted band size: 39 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?



Note: The molecular weight is larger than expected, as the recombinant protein is not the endogenouse protein, but rather the fc-fusion protein.

Overlay histogram showing SH-SY5Y cells stained with ab86501 (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 (ab86501, 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. This antibody gave a positive result in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween for 20 min used under the same conditions.