All lanes : Anti-Hsp70 antibody [5A5] (ab2787) at 1/500 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 2 : A549 (Human lung carcinoma cell line) whole cell lysate
Lane 3 : IMR32 (Human brain neuroblast cell line) whole cell lysate
Lanes 4-5 : Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 6 : MDCK (Canine kidney cell line) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate
Lane 8 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 9 : COS-7 (African green monkey kidney fibroblast-like cell line) whole cell lysate
Lane 10 : Mouse ovary tissue lysate

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : Goat anti-mouse IgG (H+L) HRP at 1/4000 dilution

Developed using the ECL technique.

Immunofluorescent analysis of U-251 MG (Human brain glioma cell line) cells labeling Hsp70 (green) with ab2787. 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 ab2787 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 normal 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 1/200 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab2787) 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.

Overlay histogram showing Jurkat cells stained with ab2787 (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 (ab2787, 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 (5min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunoprecipitation of Hsp70 was performed on HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate. Antigen:antibody complexes were formed by incubating 500µg whole cell lysate with 2µg of ab2787 overnight on a rocking platform at 4°C. The immune complexes were captured on 50µl Protein A/G 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 incubated with ab2787 (1:1000) overnight rotating at 4°C, washed in TBST, and probed with IP detection reagent-HRP at a dilution of 1:1000 for at least one hour. Chemiluminescent detection was performed.

All lanes : Anti-Hsp70 antibody [5A5] (ab2787) at 1/1000 dilution

Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate
Lane 4 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 5 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate

Lysates/proteins at 50 µg per lane.

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

Western blot analysis of Hsp70 was performed by 15µl of prestained protein ladder 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 incubated overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and incubated with secondary antibody for at least 1 hour. Chemiluminescent detection was performed.

Immunofluorescent analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Hsp70 (green) with ab2787. 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 ab2787 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 C6 (Rat glial tumor cell line) cells labeling Hsp70 (green) with ab2787. 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 (right) or with or an antibody recognizing Heat Shock Protein 70 (ab2787) (left) 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 HeLa (Human epithelial cell line from cervix adenocarcinoma) and NIH/3T3 (Mouse embryo fibroblast cell line) cells labeling Hsp70 (green) with ab2787. 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 a HSP70 Monoclonal Antibody, 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.

ab2787 staining Hsp70 in 2089 cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in methanol for 30 minutes at -20°C, washed with PBS, and incubated in blocking solution (10% human serum in PBS) for 1 hour at room temperature. Cells were stained with ab2787 diluted in blocking solution for 1 hour at room temperature in humidified chambers. Cells were washed with PBS and then incubated with secondary antibody diluted 1/200 in blocking solution for 1 hour at room temperature in opaque humidified chambers.

Immunohistochemistry was performed on cancer biopsies of deparaffinized Human prostate 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 1/100 with a mouse monoclonal antibody recognizing Heat Shock Protein 70 (ab2787) 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 normal 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 (ab2787) 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.

ab2787 staining human skin. Staining is localized to the cytoplasm and nucleus.
Left panel: with primary antibody at 1/100. Right panel: isotype control.
Sections were stained using an automated system at room temperature. Sections were rehydrated and antigen retrieved. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Western blot analysis of U2OS cell lysate (30μg/lane) labelling Hsp70 with ab2787 at 1/1000 in 5% milk in TBST for 13 hours at 4°C. A IRDye^® 800-conjugated Goat anti-Mouse polyclonal (1/10000) was used as the secondary antibody.

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