- MA3-012 - Invitrogen Antibodies HSPD1 antibody

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Higashi H, Maejima T, Lee LH, Yamazaki Y, Hottiger MO, Singh SA, Aikawa M
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Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms.

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The EMBO journal 1992 Dec;11(13):4757-65

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HSP60 Monoclonal Antibody (4B9/89)(Product # MA3-012) and a 60kDa band corresponding to HSP60 was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), Hep G2 (Lane 2), THP-1 (Lane 3), Jurkat (Lane 4), SW480 (Lane 5) and A549 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Heat Shock Protein 60 (HSP60) was performed by loading 50 µg of the indicated whole cell lysates and 15 µL 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 HSP60 monoclonal antibody (Product # MA3-012) 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 using Anti-Heat Shock Protein 60 Monoclonal Antibody (4B9/89) (Product # MA3-012) shows staining in A2058 Cells. Heat Shock Protein 60 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 60 (Product # MA3-012) at a dilution of 1:200 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 using Anti-Heat Shock Protein 60 Monoclonal Antibody (4B9/89) (Product # MA3-012) shows staining in ATDC5 Cells. Heat Shock Protein 60 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 60 (Product # MA3-012) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 using Anti-Heat Shock Protein 60 Monoclonal Antibody (4B9/89) (Product # MA3-012) shows staining in Hela Cells. Heat Shock Protein 60 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 60 (Product # MA3-012) at a dilution of 1:100 over night at 4°C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503, Goat Anti-Mouse). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 using a Hsp60 Monoclonal Antibody (Product # MA3-012) in human endothelial cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of HSP60 was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with HSP60 Monoclonal Antibody (4B9/89) (Product # MA3-012) at 1:60 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic(mitochondrial like pattern) localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 (HSP60) (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 HSP60 monoclonal antibody (Product # MA3-012), 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 60 (Hsp60, green) in HeLa 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 (left panel) or without (right panel) a Hsp60 monoclonal antibody (Product # MA3-012), 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. F-Actin (red) was stained with Dylight 554 phalloidin (Product # 21834), and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan or ToxInsight at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal deparaffinized human Kidney 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 60 (Product # MA3-012) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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 1:100 with a mouse monoclonal antibody recognizing Heat Shock Protein 60 (Product # MA3-012) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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 60 (Product # MA3-012) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of HSP60 was done on HeLa cells. Cells were fixed, permeabilized and stained with a HSP60 mouse monoclonal antibody (Product # MA3-012, orange histogram) or a mouse IgG2a isotype control (Product # MA1-10418, black histogram) at a dilution of 10 µg/mL. After incubation for 1 hour on ice, the cells were labeled with a Goat anti-Mouse IgG Secondary Antibody, DyLight 650 conjugate (Product # 84545) at a dilution of 1:50 for 1 hour on ice. A representative 10,000 cells were acquired and analyzed for each sample.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of Heat Shock Protein 60 (HSP60) was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500 µg whole cell lysate with 2 µg of HSP60 monoclonal antibody (Product # MA3-012) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL 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 HSP60 monoclonal antibody (Product # MA3-012) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with Clean-Blot IP Detection Reagent (HRP) at a dilution of 1:1000 (Product # 21230) for at least one hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4. Minocycline attenuates translation and reduces ribosomal load of highly translated mRNAs. ( A ) Autoradiograph (top) monitoring 35 S incorporation over 1 hr of HeLa cells treated for 6 hr with cycloheximide or increasing concentrations of minocycline. Coomassie gel (bottom) is shown as a loading control. Total of four independent experiments. ( B ) Polysome profile analysis of 12 hr water- (black) or minocycline-treated (blue) HeLa cells. High molecular weight polysomes ( P ) and 80S monosomes ( M ) regions are shaded in gray. An increase in monosomes and a decrease in high-molecular-weight polysomes are indicative of translation attenuation. Total of three independent experiments. ( C ) Immunoblots probing for HSP60 and HSP70 expression of water- or minocycline-treated (100 muM) HeLa cells after a 1 hr, 43degC heat shock. Total of three independent experiments. ( D ) qRT-PCR analysis of HSP60 and HSP70 mRNA expression in HeLa cells treated with or without minocycline (100 muM) before and after a 1 hr, 43degC heat shock. Minocycline treatment was initiated 12 hr prior to the heat shock. Data are represented as mean +- S.E.M. Significance determined by the Mann-Whitney t-test. Total of three independent experiments. ( E ) Immunoblots probing for BiP expression of water- or minocycline-treated (100 muM) HEK293 DAX cells engineered to allow UPR ER activation by trimethoprim treatment (TMP). Water or 100 muM minocycline was added alone or in combination with 20 uM t

MA3-012

Antibody data