All lanes : Anti-Hsp90 antibody [H90-10] (ab58950) at 0.5 µg/ml

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 - ab58950 observed at 85 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab58950 was shown to react with Hsp90 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 ab58950 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at 0.5 µg/ml 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.

Overlay histogram showing HeLa cells stained with ab58950 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab58950, 0.01μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This image was generated using the ascites version of the product. 

Western Blot analysis of the effect of siRNA inhibition of Hsp90 on INrf2. Murine hepatoma (Hepa-1) cells were transfected with control siRNA or Hsp90-specific siRNA and Hsp90 (ab58950), INrf2, and actin levels were analyzed by immunoblotting.

This image was generated using the ascites version of the product.