Immunoprecipitation of Heat Shock Protein 70 (Hsp70) was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500ug whole cell lysate with 2ug of Hsp70 monoclonal antibody (Product # MA3-006) overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Plus Agarose (Product # 20421), washed extensively, and eluted with Lane Marker Reducing Sample Buffer (Product # 39000). 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 probed with a Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).
Western blot analysis of Hsp70 was performed by loading 20ul of gill tissue lysates from the salt marsh mussel, Guekensia demissa, isolated from various coves in Rhode Island (indicated above the lanes) and processed by homogenization in 32mM Tris-HCl, 1mM EDTA, and 2% SDS homogenization buffer, per well onto an SDS-PAGE gel. Proteins extracted from the supernatants were transferred to nitrocellulose membrane and blocked overnight with 5% milk in TBST. The membrane was probed with an Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000, washed in TBST, and probed with an HRP-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:3000. Detection was performed using SuperSignal West Pico (Product # 34080). Data courtesy of the Innovators Program.
Western blot analysis of Heat Shock Protein 70 (Hsp70) was performed by loading 50ug of the indicated whole cell lysates and 15ul of PageRuler Prestained Protein Ladder (Product # 26616) 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 probed with a Hsp70 monoclonal antibody (Product # MA3-006) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween 20, and probed with a goat anti-mouse IgG-HRP secondary antibody (Product # 31430) at a dilution of 1:20,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).
Immunohistochemistry was performed on normal 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 (Product #MA3-006) 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 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 (Product #MA3-006) 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.
Immunofluorescent analysis of Heat Shock Protein 70 (Hsp70) (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 (Product # 37525) for 15 minutes at room temperature. Cells were probed with a Hsp70 monoclonal antibody (Product # MA3-006), 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 (Product # 35502) at a dilution of 1:400 for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.
Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product# MA3-006) shows 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 (Product# MA3-006) at a dilution of 1:100-1:200 over night at 4 ?C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product# 35552 for GAR, Product# 35503 for GAM). Images were taken at 60X magnification.
Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product# MA3-006) 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 (Product# MA3-006) at a dilution of 1:100-1:200 over night at 4 ?C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product# 35552 for GAR, Product# 35503 for GAM). Images were taken at 60X magnification.
Immunohistochemistry was performed on normal 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 (Product #MA3-006) 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.
Immunofluorescent analysis of Heat Shock Protein 70 using Heat Shock Protein 70 Monoclonal antibody (3A3) (Product# MA3-006) 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 (Product# MA3-006) at a dilution of 1:100-1:200 over night at 4 ?C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product# 35552 for GAR, Product# 35503 for GAM). Images were taken at 60X magnification.