Anti-Hsp90 beta抗体(ab2927)

Samples were loaded 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 then probed with primary antibody (ab2927) overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with secondary antibody for at least one hour. Chemiluminescent detection was performed using SuperSignal West Pico.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in HepG2 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Western blot of mouse HSP 86 using ab2927.

Flow cytometry analysis of HSP90 was done on HeLa cells. Cells were fixed, permeabilized and stained with a HSP90 rabbit polyclonal antibody (ab2927) (blue histogram) or a rabbit IgG isotype control (black histogram) at a dilution of 10 µg/mL. After incubation for 1 hour on ice, the cells were labeled with a Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor^® 647 conjugate at a dilution of 1/50 for 1 hour on ice. A representative 10,000 cells were acquired and analyzed for each sample.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in U251 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta (green) in HeLa and NIH3T3 cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were blocked with 1% BSA for 15 minutes at room temperature. Cells were incubated with ab2927 at a dilution of 1:50 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.

Immunocytochemistry/Immunofluorescence analysis of HSP90 beta shows staining in A2058 cells. HSP 90 beta staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or ab2927 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-rabbit secondary antibody. Images were taken at 60X magnification.

Immunoprecipitation of Hsp90 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of Hsp90 polyclonal antibody (ab2927) overnight on a rocking platform at 4°C. Immune complexes were captured on 50µl Protein A/G Agarose washed extensively and eluted with Buffer. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp90 polyclonal antibody (ab2927) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBSTand probed with HRP detection reagent at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.