All lanes : Anti-Hsp90 beta antibody [H90-10] (ab53497) at 1/5000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : HSP90AB1 knockout HEK-293T cell lysate
Lane 3 : Saos-2 cell lysate
Lane 4 : HL-60 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 83 kDa
Observed band size: 85 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab53497 observed at 85 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab53497 was shown to react with Hsp90 beta in wild-type HEK-293T cells in western blot with loss of signal observed in HSP90AB1 knockout cell line ab266117 (HSP90AB1 knockout cell lysate ab257190). Wild-type and HSP90AB1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab53497 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Ab53497 staining Human normal placenta. Staining is localized to cytoplasmic compartment.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffers EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

ICC/IF image of ab53497 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53497, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

Anti-Hsp90 beta antibody [H90-10] (ab53497) at 1 µg/ml + Recombinant human Hsp90 beta protein (Active) (ab80033) at 0.1 µg

Secondary
Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 83 kDa


Exposure time: 8 minutes

Paraffin-embedded mouse backskin (epidermis) tissue fixed with Bouin's fixative, stained for Hsp90 beta using ab53497 at 1/100 dilution in immunohistochemical analysis. Primary antibody was incubated for 1 hour at room temperature. Secondary antibody was a FITC-conjugated goat anti-mouse (green) at 1/50 dilution ncubated for 1 hour at room temperature.

Formalin-fixed, paraffin-embedded human colon carcinoma tissue stained for Hsp90 beta using ab53497 at 1/10000 in immunohistochemical analysis. Primary antibody was incubated for 12 hours at 4°C. Secondary antibody was an Alexa Fluor^® 555 goat anti-mouse (red) at 1/5000 dilution incubated for 1 hour at room temperature.

40x magnification.

ab53497 at 100,000 dilution staining Hsp90 in human colon cancer tissue section by immunohistochemistry (Formalin/ PFA fixed paraffin-embedded tissue sections). A antibody amplifier™ system was used for staining. A HRP-conjugated secondary antibody was used at 1/10 dilution

ab53497 at 100,000 dilution staining Hsp90 beta in mouse colon tissue section by immunohistochemistry (Formalin/ PFA fixed paraffin-embedded tissue sections). A antibody amplifier™ system was used for staining. An Alexa Fluor^®  568 conjugated secondary antibody was used at 1/10 dilution.

All lanes : Anti-Hsp90 beta antibody [H90-10] (ab53497)

Lane 1 : Hsp90 beta protein
Lane 2 : Hsp90 alpha protein

Lysates/proteins at 2 µg per lane.

Predicted band size: 83 kDa

Overlay histogram showing HeLa cells stained with ab53497 (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 (ab53497, 0.5µg/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 IgG2a [ICIGG2A] (ab91361, 1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.