Invitrogen

HSP60 Monoclonal Antibody (4B9/89)

Tech Support

Datasheet

Protocols

Questions & Answers

Tech Support

Product Image Gallery

HSP60 Antibody in Immunocytochemistry (ICC/IF) — 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.

HSP60 Antibody in Western Blot (WB) — 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).

HSP60 Antibody in Immunohistochemistry (Paraffin) (IHC (P)) — 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.

HSP60 Antibody in Immunoprecipitation (IP) — 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).

HSP60 Antibody in Flow Cytometry (Flow) — 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.

HSP60 Antibody — Antibody specificity was demonstrated by showing that antibodies raised against the same target protein perform similarly. Western blot of HSP60 using HSP60 Monoclonal Antibody (4B9/89) (Product # MA3-012) along with another HSP60 Polyclonal Antibody (Product # PA1-41400) shows similar expression of HSP60. {IAV}

Product Details

MA3-012

Applications	Tested Dilution	Publications
Western Blot (WB)	1:100-1:1,000	View 9 publications 9 publications
Immunohistochemistry (IHC)	1:200	View 1 publication 1 publication
Immunocytochemistry (ICC/IF)	1:50-1:60	View 2 publications 2 publications
Flow Cytometry (Flow)	1-20 µg/mL	-
ELISA (ELISA)	Assay-dependent	-
Immunoprecipitation (IP)	2 µg	-
Neutralization (Neu)	-	View 1 publication 1 publication
Miscellaneous PubMed (Misc)	-	View 1 publication 1 publication

Product Specifications
Species Reactivity	Human, Mouse, Non-human primate, Rat
Published species	Bacteria, Human, Mouse, Non-human primate, Rat
Host/Isotype	Mouse / IgG2a
Class	Monoclonal
Type	Antibody
Clone	4B9/89
Immunogen	Human placental HSP60.
Conjugate	Unconjugated Alexa Fluor 488 Alexa Fluor 555 Alexa Fluor 647
Form	Lyophilized
Concentration	1 mg/mL
Purification	Protein A
Contains	no preservative
Storage conditions	-20° C, Avoid Freeze/Thaw Cycles
RRID	AB_2121466

Product Specific Information

MA3-012 detects heat shock protein 60 kDa (HSP60) from human, mouse, rat, and primate cells, tissues and recombinant protein preparations.

MA3-012 has been successfully used in Western blot, flow cytometry, immunocytochemistry and ELISA procedures. By Western blot, this antibody detects a single 58 kDa protein representing HSP60 from human blood samples. Immunocytochemical staining of HSP60 in B-SC-1 cells with MA3-012 yields a pattern consistent with the mitochondrial localization of HSP60.

The MA3-012 antigen is human placental HSP60. Epitope mapping studies using human HSP60 deletion mutants suggest that this antibody binds either between amino acids 335-366 or 484-547.

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.

Reconstitute in 100 µL PBS to create a stock of 1 mg/mL.

Target Information

This gene encodes a member of the chaperonin family. The encoded mitochondrial protein may function as a signaling molecule in the innate immune system. This protein is essential for the folding and assembly of newly imported proteins in the mitochondria. This gene is adjacent to a related family member and the region between the 2 genes functions as a bidirectional promoter. Several pseudogenes have been associated with this gene. Two transcript variants encoding the same protein have been identified for this gene. Mutations associated with this gene cause autosomal recessive spastic paraplegia 13.

For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.

Product References

The Role of Nonglycolytic Glucose Metabolism in Myocardial Recovery Upon Mechanical Unloading and Circulatory Support in Chronic Heart Failure.

Circulation

Badolia R,Ramadurai DKA,Abel ED,Ferrin P,Taleb I,Shankar TS,Krokidi AT,Navankasattusas S,McKellar SH,Yin M,Kfoury AG,Wever-Pinzon O,Fang JC,Selzman CH,Chaudhuri D,Rutter J,Drakos SG

MA3-012 was used in Western Blotting to provide further insights into mechanisms responsible for the beneficial effect of glycolysis induction during the recovery of failing human hearts after mechanical unloading.

Tue Jul 21 00:00:00 EDT 2020

A Study into the ADP-Ribosylome of IFN-γ-Stimulated THP-1 Human Macrophage-like Cells Identifies ARTD8/PARP14 and ARTD9/PARP9 ADP-Ribosylation.

Journal of proteome research

Higashi H,Maejima T,Lee LH,Yamazaki Y,Hottiger MO,Singh SA,Aikawa M

MA3-012 was used in Western Blotting to identify interferon-γ-induced ADP-ribosylation substrates in macrophages.

Fri Apr 05 00:00:00 EDT 2019

Translation attenuation by minocycline enhances longevity and proteostasis in old post-stress-responsive organisms.

eLife

Solis GM,Kardakaris R,Valentine ER,Bar-Peled L,Chen AL,Blewett MM,McCormick MA,Williamson JR,Kennedy B,Cravatt BF,Petrascheck M

Published figure using HSP60 monoclonal antibody (Product # MA3-012) in Western Blot

Tue Nov 27 00:00:00 EST 2018

Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT.

Nature communications

Schueder F,Lara-Gutiérrez J,Beliveau BJ,Saka SK,Sasaki HM,Woehrstein JB,Strauss MT,Grabmayr H,Yin P,Jungmann R

MA3-012 was used in Immunocytochemistry to combine 3D DNA points accumulation for imaging in nanoscale topography with spinning disk confocal hardware to remove the requirement of complex hardware for single-molecule localisation microscopy in thick samples.

Tue Dec 12 00:00:00 EST 2017

Fancd2 in vivo interaction network reveals a non-canonical role in mitochondrial function.

Scientific reports

Zhang T,Du W,Wilson AF,Namekawa SH,Andreassen PR,Meetei AR,Pang Q

MA3012 was used in western blot to explore the role of Fancd2 in normal development and tumorigenesis

Wed Apr 05 00:00:00 EDT 2017

CCN6 regulates mitochondrial function.

Journal of cell science

Patra M,Mahata SK,Padhan DK,Sen M

MA3-012 was used in western blot to determine regulation of mitochondrial function via a Wnt induced signaling protein-3 called CCN6

Fri Jul 15 00:00:00 EDT 2016

Adipocytes from New Zealand obese mice exhibit aberrant proinflammatory reactivity to the stress signal heat shock protein 60.

Journal of diabetes research

Märker T,Kriebel J,Wohlrab U,Burkart V,Habich C

MA3-012 was used in Binding experiments to study whether adipocytes of diabetes-predisposed mice exhibit an increased proinflammatory reactivity to Hsp60.

Tue Oct 28 00:00:00 EDT 2014

Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase.

The Journal of biological chemistry

Ahuja N,Schwer B,Carobbio S,Waltregny D,North BJ,Castronovo V,Maechler P,Verdin E

MA3-012 was used in western blot to study the regulation of insulin secretion by SIRT4

Fri Nov 16 00:00:00 EST 2007

Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.

The Journal of biological chemistry

Osterloh A,Kalinke U,Weiss S,Fleischer B,Breloer M

MA3-012 was used in blocking/activating experiment to investigate the HSP60-mediated immune responses and its relation with lipopolysaccharide

Fri Feb 16 00:00:00 EST 2007

The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.

The Journal of cell biology

Schwer B,North BJ,Frye RA,Ott M,Verdin E

MA3-012 was used in western blot to characterize a new mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase .

Mon Aug 19 00:00:00 EDT 2002

The identification of secreted heat shock 60 -like protein from rat glial cells and a human neuroblastoma cell line.

Neuroscience letters

Bassan M,Zamostiano R,Giladi E,Davidson A,Wollman Y,Pitman J,Hauser J,Brenneman DE,Gozes I

MA3-012 was used in western blot to study the effect of vasoactive intestinal peptide and temperature on hsp60 expression in glial and neuroblastoma cells

Fri Jun 26 00:00:00 EDT 1998

Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury.

Circulation

Lau S,Patnaik N,Sayen MR,Mestril R

MA3-012 was used in immunocytochemistry to investigate the protective effect of HSP60 and HSP10 on myogenic cells against ischemia/reperfusion injury

Tue Oct 07 00:00:00 EDT 1997

Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells.

Experimental cell research

Soltys BJ,Gupta RS

MA3-012 was used in immunohistochemistry to investigate the localization of the 60-kDa heat shock chaperonin protein

Wed Jan 10 00:00:00 EST 1996

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

The EMBO journal

Langer T,Pfeifer G,Martin J,Baumeister W,Hartl FU

MA3-012 was used in western blot to investigate the important role of GroEL in protein folding

Tue Dec 01 00:00:00 EST 1992

Bioinformatics

Protein Aliases: 60 kDa chaperonin; 60 kDa heat shock protein; 60 kDa heat shock protein, mitochondrial; Chaperonin 60; CPN60; heat shock 60kD protein 1 (chaperonin); heat shock 60kDa protein 1; heat shock 60kDa protein 1 (chaperonin); Heat shock protein; Heat shock protein 60; heat shock protein 60 (liver); heat shock protein 65; heat shock protein, 60 kDa; HSP; HSP-60; HSP-65; Hsp60s1; HuCHA60; Mitochondrial matrix protein P1; P60 lymphocyte protein; short heat shock protein 60 Hsp60s1

Gene Aliases: 60kDa; CPN60; GROEL; HLD4; HSP-60; HSP60; HSP65; HSPD1; Hspd1-30p; HuCHA60; SPG13

UniProt ID: (Human) P10809, (Mouse) P63038, (Rat) P63039

Entrez Gene ID: (Human) 3329, (Mouse) 15510, (Rat) 63868

Function(s)

Process(es)

lipopolysaccharide binding p53 binding DNA replication origin binding single-stranded DNA binding double-stranded RNA binding protein binding ATP binding ATPase activity ubiquitin protein ligase binding poly(A) RNA binding unfolded protein binding chaperone binding nucleotide binding protease binding protein complex binding insulin binding protein heterodimerization activity misfolded protein binding

HSP60 Monoclonal Antibody (4B9/89)

Product Details

MA3-012

Western Blot (WB)

Immunohistochemistry (IHC)

Immunocytochemistry (ICC/IF)

Flow Cytometry (Flow)

ELISA (ELISA)

Immunoprecipitation (IP)

Neutralization (Neu)

Miscellaneous PubMed (Misc)

Species Reactivity

Published species

Host/Isotype

Class

Type

Clone

Immunogen

Conjugate

Form

Concentration

Purification

Contains

Storage conditions

RRID

Product Specific Information

Target Information

Bioinformatics

Performance Guarantee

We're here to help

Your items have has been added!