All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1/1000 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
Lane 4 : Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate
Lane 5 : Jurkat whole cell lysate treated with heat shock (42deg for 30 min followed by 37deg for 3 hours)
Lane 6 : MDBK (bovine kidney cell line) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic 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

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

Predicted band size: 70-78 kDa

Immunofluorescence analysis of HEK-293 (human epithelial cell line from embryonic kidney) cells labeling HSP70 with ab5439 at 1/200 dilution in 0.1% BSA, incubated at 4°C overnight, followed by Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant 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 localization to nucleus and cytoplasm. 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 2% BSA for 1 hour at room temperature. The images were captured at 60X magnification.

All lanes : Anti-Hsp70 antibody [3A3] (ab5439) at 1 µg/ml

Lane 1 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast cell line) whole cell lysate
Lane 4 : F9 (mouse embryonic testicular cancer cell line) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 70-78 kDa
Observed band size: 73 kDa why is the actual band size different from the predicted?

Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) ab5439 shows staining in MCF7 (human breast adenocarcinoma cell line) 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 (human epithelial cell line from cervix adenocarcinoma) 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 (mouse embryonic fibroblast cell line) 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 (human epithelial cell line from cervix adenocarcinoma) 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 (human T cell leukemia cell line from peripheral blood) 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.