Immunohistochemistry analysis of ab59459 staining Hsp90 alpha in mouse inflamed colon tissue section at 1/100000 dilution for 12 hours at 4°C using antibody amplifier™ system. Biotin Goat Anti-Mouse antibody was used at 1:2000 for 1 hour at room tempertature. Mayer Hematoxylin (purple/blue) nuclear stain at 200 µl for 2 minutes at room temperature was used as a counter stain. Localization: Inflammatory cells. Magnification: 40x.

All lanes : Anti-Hsp90 antibody [D7a] (ab59459) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : HSP90AA1 knockout HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : HAP1 whole cell lyate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) at 1/10000 dilution

Predicted band size: 95 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab59459 observed at 90 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab59459 Anti-Hsp90 antibody [D7a] was shown to specifically react with Hsp90 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266591 (knockout cell lysate ab258458) was used. Wild-type and Hsp90 knockout samples were subjected to SDS-PAGE. ab59459 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab59459 staining Hsp90 alpha in human colon cancer tissue section by immunohistochemistry (Bouin's fixed paraffin embedded tissue sections). Tissue underwent heat mediated antigen retrieval in microwave in citrate buffer. The primary antibody was used at dilution of 1/100,000 dilution using antibody amplifier™ system.

Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: Hsp90 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: Hek 293 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab59459 observed at 90 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab59459 was shown to specifically recognize Hsp90 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when Hsp90 knockout cells were examined. Wild-type and Hsp90 knockout samples were subjected to SDS-PAGE. Ab59459 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20,000 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 1/20,000 dilution for 1 hour at room temperature before imaging.