All lanes : Anti-Hsp60 antibody [2E1/53] (ab5479) at 1/2000 dilution

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : A431 (human epidermoid carcinoma cell line) whole cell lysate
Lane 3 : HEK-293 (h
uman epithelial cell line from embryonic kidney) whole cell lysate
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 5 : COS-7 (african green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 6 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
Lane 7 : THP1 (human monocytic leukemia cell line) whole cell lysate
Lane 8 : K562 (human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate
Lane 9 : A549 (human lung carcinoma cell line) whole cell lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate at 1/4000 dilution

Immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling HSP60 at 1/200 dilution in 0.1% BSA, incubated at 4°C overnight, followed by Goat anti-Mouse IgG (H+L)/IgM (L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at 1/2000 dilution for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c: red) was stained with Rhodamine Phalloidin at 1/300 dilution. Panel d represents the merged image showing mitochondrial localization. Panel e represents control cells with no primary antibody to assess background. 

The cells (70% confluent log phase) were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The images were captured at 60X magnification.

All lanes : Anti-Hsp60 antibody [2E1/53] (ab5479) at 1 µg/ml (overnight at 4°C)

Lane 1 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 2 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 3 : K562 (human chronic myelogenous leukemia lymphoblast cell line ) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 4 : A431 (human epidermoid carcinoma cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 5 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 6 : U-2 OS (human bone osteosarcoma epithelial cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 7 : COS-7 (african green monkey kidney fibroblast-like cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 8 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour
Lane 9 : NRK (rat kidney cell line) whole cell lysate with 5% BSA/TBST for at least 1 hour

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : Goat anti-mouse IgM secondary antibody at 1/20000 dilution

Western blot analysis of various cell lines (50µg/lane) labeling Hsp60 with ab5479 at 1µg/ml.

Immunofluorescence analysis of Hsp60 (green) in HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp60 with ab5479 at 1/200 dilution and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI.

Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Images were taken at a 60X magnification.

Immunofluorescence analysis of A431 (human epidermoid carcinoma cell line) cells labeling Hsp60 (green) with ab5479 at 1/100 dilution and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. 

Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Images were taken at a 60X magnification.

Immunofluorescence analysis of NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling Hsp60 (green) with ab5479 at 1/20 dilution and incubated overnight in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody for 45 minutes at room temperature in the dark. F-actin (red) was stained with a fluorescent phalloidin and nuclei (blue) were stained with DAPI. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes at room temperature and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with Images were taken at a 60X magnification.

Immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp60 (green) with ab5479 at 1/100 dilution or without overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. 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. Images were taken at 60X magnification.

Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Hsp60 (green) with ab5479 at 1/100 dilution or without (negative control) overnight at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. 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. Images were taken at 60X magnification.

Immunofluorescence analysis of U-251 MG (formally U-373 MG) (human brain glioma cell line) cells labeling Hsp60 with ab5479 at 1/200 dilution overnight at 4°C, washed with PBS, followed by DyLight-488 conjugated secondary antibody.

Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with ab5479. Heat Shock Protein 60 (Hsp60) staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification.

Immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) and NIH/3T3 (mouse embryonic fibroblast cell line) cells labeling HSP60 (green) with ab5479 at 10ug/ml for at least 1 hour at room temperature. Cells were washed with PBS and incubated with a fluorescently labeled goat anti-mouse IgM secondary antibody at 1/400 dilution for 30 minutes at room temperature. F-Actin (red) was stained with DyLight 554 Phalloidin and nuclei (blue) were stained with Hoechst 33342 dye.

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. Images were taken at 20X magnification.

Immunoprecipitation of Hsp60 was performed on HeLa (human epithelial cell line from cervix adenocarcinoma) cells. Antigen-antibody complexes were formed by incubating 500ug of whole cell lysate with 2ug of ab5479 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 5X Lane Marker Reducing Sample Buffer. Samples were 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 ab5479 at a concentration of 1ug/ml overnight rotating at 4°C, washed in TBST, and probed with a goat anti-mouse IgM secondary antibody at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection performed using SuperSignal West Dura.

Immunohistochemiscal analysis of both normal and cancer biopsies of deparaffinized human breast carcinoma tissues labeling Hsp60 with ab5479 at 1/20 dilution 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.

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. 

Immunohistochemiscal analysis of both normal and cancer biopsies of deparaffinized human kidney tissue labeling Hsp60 with 1/100 dilution 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.

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.