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.
|Tested species reactivity||Human, Mouse, Non-human primate, Rat|
|Published species reactivity||Rat, Non-human primate, Bacteria, Human, Mouse, Not Applicable|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||Human placental HSP60.|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay dependent|
|Flow Cytometry (Flow)||1-20 µg/ml|
|Immunocytochemistry (ICC)||10 µg/ml|
|Western Blot (WB)||1:100 - 1:1000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
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 with PBS.
Heat shock proteins (HSP) are expressed in response to various biological stresses, including high temperatures. There are several major families of HSPs including HSP70, HSP90 and HSP100. HSP60, an ~60 kDa protein, is the human homologue of HSP65 (a major antigenic mycobacterial protein) and E. coli GroEL. HSP60, also referred to as chaperonin-60, P1 and mitonin, has been classified as a member of a family of proteins termed chaperonins which act to recognize and stabilize polypeptide intermediates during folding, assembly and disassembly. HSP60 is a constitutively expressed mitochondrial protein that shares many characteristics with HSP70 including high abundance, induction by environmental stress, ATPase activity and a critical role in protein assembly and folding. However, in contrast with HSP70, HSP60 functions as a 14 subunit oligomer which is thought to interact with only 1 or 2 target polypeptides at one time.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
|Not Applicable||Not Cited||
CCN6 regulates mitochondrial function.
MA3-012 was used in western blot to determine regulation of mitochondrial function via a Wnt induced signaling protein-3 called CCN6
|Patra M,Mahata SK,Padhan DK,Sen M||Journal of cell science (129:2841)||2016|
Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase.
MA3-012 was used in western blot to study the regulation of insulin secretion by SIRT4
|Ahuja N,Schwer B,Carobbio S,Waltregny D,North BJ,Castronovo V,Maechler P,Verdin E||The Journal of biological chemistry (282:33583)||2007|
The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase.
MA3-012 was used in western blot to characterize a new mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase .
|Schwer B,North BJ,Frye RA,Ott M,Verdin E||The Journal of cell biology (158:647)||2002|
The identification of secreted heat shock 60 -like protein from rat glial cells and a human neuroblastoma cell line.
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
|Bassan M,Zamostiano R,Giladi E,Davidson A,Wollman Y,Pitman J,Hauser J,Brenneman DE,Gozes I||Neuroscience letters (250:37)||1998|
Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.
MA3-012 was used in western blot to investigate the important role of GroEL in protein folding
|Langer T,Pfeifer G,Martin J,Baumeister W,Hartl FU||The EMBO journal (11:4757)||1992|
Synergistic and differential modulation of immune responses by Hsp60 and lipopolysaccharide.
MA3-012 was used in blocking/activating experiment to investigate the HSP60-mediated immune responses and its relation with lipopolysaccharide
|Osterloh A,Kalinke U,Weiss S,Fleischer B,Breloer M||The Journal of biological chemistry (282:4669)||2007|
Simultaneous overexpression of two stress proteins in rat cardiomyocytes and myogenic cells confers protection against ischemia-induced injury.
MA3-012 was used in immunocytochemistry to investigate the protective effect of HSP60 and HSP10 on myogenic cells against ischemia/reperfusion injury
|Lau S,Patnaik N,Sayen MR,Mestril R||Circulation (96:2287)||1997|
|Non-human primate||Not Cited||
Immunoelectron microscopic localization of the 60-kDa heat shock chaperonin protein (Hsp60) in mammalian cells.
MA3-012 was used in immunohistochemistry to investigate the localization of the 60-kDa heat shock chaperonin protein
|Soltys BJ,Gupta RS||Experimental cell research (222:16)||1996|