Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker

• WB

Lab

Western blot - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

Lanes 1 - 4 : Merged signal (red and green). Green - ab86472 observed at 40 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab86472 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. ab86472 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.

All lanes:

Western blot - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (ab86472) at 1/500 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

ISL1 (Islet 1) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

SH-SY5Y whole cell lysate at 20 µg

Lane 4:

HepG2 whole cell lysate at 20 µg

Predicted band size: 39 kDa

Observed band size: 40 kDa

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• Flow Cyt

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Flow Cytometry - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

Overlay histogram showing SH-SY5Y cells stained with ab86472 (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 (ab86472, 1/50 dilution) 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 IgG1 [ICIGG1] (ab91353, 2μg/1x10^6 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

ab86472 at a 1/200 dilution staining Islet 1 in human lung cancer tissue. DAB staining shows nuclear localisation.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

Confocal immunofluorescence analysis of HEK293 cells trasfected with full-length Islet 1-hIgGFc using ab86472 at 1/150 dilution (green). Blue : DRAQ5 fluorescent DNA dye.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

ab86472 at a 1/200 dilution staining Islet 1 in human cervical carcinoma tissue. DAB staining shows nuclear localization.

• WB

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Western blot - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (AB86472)

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

All lanes:

Western blot - Anti-Islet 1 antibody [1H9] - Neural Stem Cell Marker (ab86472) at 1/500 dilution

All lanes:

HEK293 cells transfected with full length Islet 1 (aa1-349)-hIgGFc

Predicted band size: 39 kDa

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