MA3-015

antibody from Invitrogen Antibodies

Targeting: HSPB1 CMT2F, Hs.76067, Hsp25, HSP27, HSP28

Provider product page for MA3-015

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Immunoelectron microscopy

Other assay

Antibody data

Antibody Data
Antigen structure
References [39]
Comments [0]
Validations
Western blot [3]
Immunocytochemistry [5]
Immunohistochemistry [3]
Other assay [2]

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Product number: MA3-015 - Provider product page
Provider: Invitrogen Antibodies
Product name: HSP27 Monoclonal Antibody (G3.1)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-015 detects heat shock protein 27 kDa (HSP27) from human, rat, mouse, monkey, and canine samples. MA3-015 has been successfully used in Western blot, immunohistochemistry (paraffin), immunoprecipitation, immunofluorescence, immunocytochemistry and ELISA procedures. By Western blot, this antibody detects an ~24 kDa protein representing HSP27 in MCF-7 cell extract. Immunohistochemical staining of human cervix with MA3-015 results in intense cytoplasmic staining of the epithelium within the parabasal cell layer. Western blot detection of HSP27 in canine heart samples is weak and may require optimization. The MA3-015 antigen is partially purified HSP27 derived from MCF-7 cytosol. The mouse HSP27 sequence is strongly conserved within the human sequence, so cross-reactivity with mouse HSP27 is expected. Antibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay.
Reactivity: Human, Rat, Canine
Host: Mouse
Isotype: IgG
Antibody clone number: G3.1
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

HSPB1 protein structure - MA3-015 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Heat Shock Protein 27 (HSP27) was performed by loading 50 µg of the indicated whole cell lysates per well 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 HSP27 monoclonal antibody (Product # MA3-015) 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: Knockout of HSP27 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR990059_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HSP27 was performed by loading 30 µg of HeLa Wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa HSP27 KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HSP27 Monoclonal Antibody (G3.1) (Product # MA3-015, 1:1,000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to HSP27.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HSP27 Monoclonal Antibody (G3.1)(Product # MA3-015) and a 25kDa band corresponding to HSP27 was observed across cell lines tested except HT-1080 which is reported to be negative. Whole cell extracts (30 µg lysate) of MCF7 (Lane 1), MDA-MB-231 (Lane 2), PC-3 (Lane 3), DU 145 (Lane 4), HeLa (Lane 5), LNCaP (Lane 6) and HT-1080 (Lane 7) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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: Immunofluorescent analysis of Heat Shock Protein 27 using Anti-Heat Shock Protein 27 Monoclonal Antibody (G3.1) (Product # MA3-015) shows staining in C6 Cells. Heat Shock Protein 27 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 27 (Product # MA3-015) at a dilution of 1:20 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 27 using Anti-Heat Shock Protein 27 Monoclonal Antibody (G3.1) (Product # MA3-015) shows staining in Hela Cells. Heat Shock Protein 27 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 27 (Product # MA3-015) 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 27 using Anti-Heat Shock Protein 27 Monoclonal Antibody (G3.1) (Product # MA3-015) shows staining in U251 Cells. Heat Shock Protein 27 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 27 (Product # MA3-015) 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 27 (HSP27) (green) in HeLa cells and negative control 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 HSP27 monoclonal antibody (Product # MA3-015), 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 at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Heat Shock Protein 27 (Hsp27, 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 Hsp27 monoclonal antibody (Product # MA3-015), at a dilution of 1:100 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 cancer biopsies of deparaffinized Human breast carcinoma tissues. 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 1:500 with a mouse monoclonal antibody recognizing Heat Shock Protein 27 (Product # MA3-015) 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 colon carcinoma tissues. 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 27 (Product # MA3-015) 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 tissues. 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 1:500 with a mouse monoclonal antibody recognizing Heat Shock Protein 27 (Product # MA3-015) 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: Immunoprecipitation of Heat Shock Protein 27 (HSP27) was performed on HeLa cells. Antigen:antibody complexes were formed by incubating 500 µg whole cell lysate with 2 µg of HSP27 monoclonal antibody (Product # MA3-015) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). HeLa cell lysate (25 µg) was loaded as a positive control. 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 a HSP27 monoclonal antibody (Product # MA3-015) at a dilution of 1:1000 overnight rotating at 4°C, washed in TBST, and probed with Clean-Blot IP Detection Reagent-HRP (Product # 21230) at a dilution of 1:1000 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: Fig. 3 AR interacts with and is a substrate for the SUMO E3 ligase Hsp27. a Ectopic induction of a hyperSUMO environment in MCF-7 cells with HA-SUMO3 increases the interaction between AR and Hsp27. b AR-Hsp27 interaction complex is enhanced endogenously in the naturally occurring hyperSUMO environment of TamR-7 cells. a - b Interactions with chromatin-bound (CB) AR were analyzed by western blot. c Immunoprecipitation of Hsp27 was performed to assess its binding with AR, Ubc9 and SUMO-2/3 in a cell-free system. d SUMOylation of AR is enhanced under in vitro conditions when recombinant Hsp27 is added. Details are found in (Additional file 1 )