Anti-Hsp60抗体[4B9/89] (ab5478)

Immunocytochemistry/Immunofluorescence analysis of Hsp60 in A2058 Cells. Hsp60 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 ab5478 at a dilution of 1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse IgG secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Hsp60 in ATDC5 Cells. Hsp60 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 incubated without (control) or with ab5478 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse IgG secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Hsp60 in Hela Cells. Hsp60 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 incubated without (control) or with ab5478 at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated goat anti-mouse secondary antibody. Images were taken at 60X magnification.

Immunocytochemistry/Immunofluorescence analysis of Hsp60 (green) in HeLa cells. 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 (left panel) or without (right panel) ab5478 at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-mouse IgG secondary antibody at a dilution of 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. Images were taken at 20X magnification.

Flow cytometry analysis of HSP60 was done on HeLa cells. Cells were fixed, permeabilized and stained with a HSP60 mouse monoclonal antibody (orange histogram) or a mouse IgG2a 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-Mouse IgG Secondary Antibody, DyLight 650 conjugate at 1/50 dilution for 1 hour on ice. A representative 10,000 cells were acquired and analyzed for each sample.

Immunocytochemistry/Immunofluorescence analysis of Hsp60 (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 and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with ab5478 at a dilution of 1:50 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat-anti-mouse IgG secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye. Images were taken at 20X magnification.

Immunoprecipitation of Hsp60 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of HSP60 monoclonal antibody (ab5478) overnight on a rocking platform at 4°C. The immune complexes were captured on 50µl Protein Agarose washed extensively and eluted with Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel then transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a HSP60 monoclonal antibody (ab5478) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBSTand probed with Detection Reagent (HRP) at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.