Anti-Hsp90 alpha antibody (ab2928)

Western blot analysis of HSP90 alpha was performed by loading 50ug of the indicated whole cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with ab2928 at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a goat anti-rabbit IgG HRP secondary antibody (1:20,000) for at least 1 hour. Chemiluminescent detection was performed.

ab2928 labelling Hsp90 alpha in Human colon adenocarcinoma tissue sections by Immunohistochemistry (formalin/PFA-fixed paraffin-embedded sections). To expose target proteins, heat-induced epitope retrieval was performed using 10mM sodium citrate (pH 6.0) buffer for 20 minutes at 95ºC. Tissues were blocked in 3% BSA in PBST for 30 minutes at room temperature. Tissue sections were incubated with the primary antibody (1:100) for 1 hour. A HRP-conjugated goat anti-rabbit IgG (1:250) was used as the secondary antibody, followed by colorimetric detection using Metal Enhanced DAB Substrate Kit. Tissues were counterstained with hematoxylin and prepped for mouting. Images were taken at 40X magnification.

Immunocytochemistry/Immunofluorescence analysis of HSP90 alpha (green) in HeLa cells and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with ab2928 at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488 goat-anti-rabbit IgG secondary antibody (1:400) for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

Immunoprecipitation of HSP90 alpha was performed on HeLa cells. Antigen-antibody complexes formed by incubating 500ug whole cell lysate with 2ug of ab2928 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Plus Agarose, washed extensively, and eluted with 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 ab2928 at a dilution of 1:1000 overnight rotating at 4°C. The membrane was washed in TBST, and probed with detection reagent at a dilution of 1:1000 for at least 1 hour. Chemiluminescent detection was performed.

Western blot of mouse Hsp90 alpha and beta using both ab2928 and ab2927 respectively.