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Our Abpromise guarantee covers the use of ab5581 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC||1/50 - 1/200.|
|IHC-P||1/50 - 1/200.|
|WB||1/100 - 1/1000. Predicted molecular weight: 23 kDa.Can be blocked with Human Hsp27 (phospho S15) peptide (ab41772).|
|ICC/IF||1/50 - 1/200.|
|IP||Use at an assay dependent concentration.
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.
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