All lanes : Anti-Insulin degrading enzyme / IDE antibody [EPR6099] (ab109538) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IDE knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 118 kDa
Observed band size: 118 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab109538 observed at 118 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109538 was shown to react with Insulin degrading enzyme / IDE in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab261755 (knockout cell lysate ab257197) was used. Wild-type HeLa and IDE knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109538 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling Insulin degrading enzyme / IDE with purified ab109538 at 1/150 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti-rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

All lanes : Anti-Insulin degrading enzyme / IDE antibody [EPR6099] (ab109538) at 1/2000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : IDE knockout HAP1 cell lysate
Lane 3 : K562 cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 118 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab109538 observed at 118 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109538 was shown to specifically react with IDE in wild-type HAP1 cells. No band was observed when IDE knockout samples were examined. Wild-type and IDE knockout samples were subjected to SDS-PAGE. ab109538 and ab8245 (loading control to GAPDH) were both diluted 1/2000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-Insulin degrading enzyme / IDE antibody [EPR6099] (ab109538) at 1/10000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : HepG2 cell lysate
Lane 3 : A375 cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 118 kDa