Anti-Hsp27 (phospho S15) antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Hsp27 (phospho S15) antibody (AB5581)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - 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.

• IP

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Immunoprecipitation - Anti-Hsp27 (phospho S15) antibody (AB5581)

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.

All lanes:

Immunoprecipitation - Anti-Hsp27 (phospho S15) antibody (ab5581)

Predicted band size: 23 kDa

false

• WB

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Western blot - Anti-Hsp27 (phospho S15) antibody (AB5581)

All lanes:

Western blot - Anti-Hsp27 (phospho S15) antibody (ab5581) at 1/500 dilution

Lane 1:

HeLa cell lysate - untreated at 50 µg

Lane 2:

HeLa cell lysate - treated with 10nM Anisomycin at 50 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/20000 dilution

Predicted band size: 23 kDa

false