Anti-Hsp70 antibody [3A3] (ab5439)

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Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in MCF-7 cells. Heat Shock Protein 70 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 an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4 ^oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439shows staining in HeLa cells. Heat Shock Protein 70 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 an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4^oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in NIH-3T3 cells. Heat Shock Protein 70 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 an antibody recognizing Heat Shock Protein 70 ab5439 at a dilution of 1/100-1/200 over night at 4^oC washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Breast carcinoma tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/50 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Testis tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunohistochemistry was performed on both normal and cancer biopsies of deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1/20 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab5439) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Immunoprecipitation of Hsp70 was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500ug whole cell lysate with 2ul of Hsp70 monoclonal antibody (ab5439) overnight on a rocking platform at 4^oC. The immune complexes were captured on 50ul Protein Agarose washed extensively and eluted with Buffer. Samples were then resolved on a 4-20% Tris-HCl polyacrylamide gel and transferred to a PVDF membraneand blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a Hsp70 monoclonal antibody (ab5439) at a dilution of 1/1000 overnight rotating at 4^oC and probed with goat anti-mouse IgG-HRP secondary antibody at a dilution of 1/20,000 for at least 1 hour. Chemiluminescent detection was performed.

Overlay histogram showing Jurkat cells stained with ab5439 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab5439, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.