Anti-Hsp27 (phospho S15) antibody (ab5581)

Immunocytochemistry analysis of Hsp27 using ab5581 at 2µg/mL concentration shows staining in 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa Cells treated with Anisomysin. The secondary antibody was Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate. Hsp27 (green), F-Actin staining with Alexa Fluor® 555 Rhodamine Phalloidin (red) and nuclei with SlowFade® Gold Antifade Mountant with DAPI (blue) is shown. Negative control has no primary antibody.

Immunoprecipitation of Hsp27 (phospho S15) was performed on HeLa cells treated with 10uM Anisomysin for 30 minutes. Antigen-antibody complexes were formed by incubating 500ug of whole cell lysate with 3ug of ab5581 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab5581 at 1:500 overnight rotating at 4°C, washed with TBST, and probed with Clean-Blot IP Detection Reagent at 1/1000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.

Immunocytochemistry/Immunofluorescence analysis of Hsp27 (phospho S15) (green) in HeLa cells either untreated (left) or treated with 10uM Anisomysin (right) for 30 minutes. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% blocker BSA for 15 minutes at room temperature. Cells were incubated with ab5581 at 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye. 20X magnification.