All lanes : Anti-Hsp90 alpha antibody [2G5.G3] (ab79849) 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: 85 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 - ab79849 observed at 90 kDa. Red - loading control ab181602 observed at 36 kDa.

 ab79849 Anti-Hsp90 alpha antibody [2G5.G3]  was shown to specifically react with Hsp90 alpha 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 alpha knockout samples were subjected to SDS-PAGE. ab79849 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.

IHC image of Hsp90 alpha staining in Human Testis 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 ab79849, 0.5 µg/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.

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

Lanes 1 - 3: Merged signal (red and green). Green - ab79849 observed at 90 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab79849 was shown to specifically react with Hsp90 alpha in wild-type HAP1 cells as signal was lost in Hsp90 alpha knockout cells. Wild-type and Hsp90 alpha knockout samples were subjected to SDS-PAGE. Ab79849 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 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.

 

ab79849 at 1/100 dilution staining Hsp90 alpha in human keratinocyte cell line HaCaT by Immunocytochemistry/ immunofluorescence. A Fluorophore conjugated goat anti mouse was used as secondary at 1/50 dilution.

Overlay histogram showing HeLa cells stained with ab79849 (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 (ab79849, 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.