Anti-Hsp27 (phospho S85)抗体(ab5594)

Immunohistochemistry analysis of paraffin-embedded human skeletal muscle tissue labelling Hsp27 (phospho S85) with ab5594 (right) compared to a negative control without primary antibody (left).

To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab5594 diluted in 3% BSA-PBS at 1/100 dilution for 1 hour at 37°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Immunocytochemistry/Immunofluorescence analysis of Hsp27 (phospho S85) (green) in HeLa cells either left untreated (left panel) or treated with 10uM Anisomysin (right panel) 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 probed with ab5594 at 20ug/ml for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody at 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.

Immunoprecipitation of Hsp27 (phospho S85) was performed on HeLa cells treated with 10uM Anisomysin for 30 minutes. Antigen-antibody complexes were formed by incubating 500µg of whole cell lysate with 3µg of ab5594 overnight on a rocking platform at 4°C. The immune complexes were captured on 50µl Protein A/G Agarose, washed extensively, and eluted with Lane Marker Reducing Sample Buffer. HeLa cell lysate (50ug) was loaded as a positive control (left lane). Samples were 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 ab5594 at 2ug/ml overnight rotating at 4°C, washed in TBST, and probed with Clean-Blot IP Detection Reagent at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura.