1.3X105 HEL cells were lysed by the following lysis buffer: 10mM Tris, pH 7.4, 150mM NaCl, 1mM CaCl2, 1mM MgCl2, 0.02% NaN3, 1mM PMSF, 0.5 lg/ml leupeptin, 1 lg/ml pepstatin A, and 10 KIU/ml aprotinin containing 1% detergent (Brij 97 or Triton). The lysate was separated by SDS/PAGE (5–15% gel) under non-reducing conditions and transferred to a PVDF membrane. ab59509 incubation time was 1h. 1,3 10⁵ HEL cells were lysed (lysis buffer containing 1%Brij97 or triton) Migration SDS/page 5-15% gel under non-reducing conditions.

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

Overlay histogram showing HeLa cells stained with ab59509 (red line). The cells were fixed with 80% methanol (5 min) and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab59509, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was a goat anti-mouse DyLight® 488 (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.

Please note that Abcam do not have any data for use of this antibody on non-fixed cells. We welcome any customer feedback.

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