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Heat Shock 60kDa Protein 1 (Chaperonin) (HSPD1) ELISA Kit
|9 references available|
|Price||379.50 $ Plus shipping costs $45.00|
|Availability||Will be delivered in 2 to 3 Business Days|
|Method type||Sandwich ELISA|
|Alternative name||Heat Shock Protein-60 (Hsp60)|
|Sample Volume||50 µL|
|Assay Time||less than 5 hours|
|Components||Human Hsp60 Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human Hsp60. Sealing Tapes: Each kit contains 3 pre-cut, pressure-sensitive sealing tapes that can be cut to fit the format of the individual assay. Human Hsp60 Standard: Human Hsp60 in a buffered protein base (40 ng, lyophilized, 2 bottles). Biotinylated Hsp60 Antibody (70x): A 70-fold concentrated biotinylated polyclonal antibody against Hsp60 (105µl). EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (20 ml). Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 ml, 2 bottles). Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrated (80µl). Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 ml). Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 ml).|
|Description||Heat-shock protein of 60 kDa (Hsp60) is a mitochondrial chaperonin involved in folding, assembly, and transport of newly imported protein from cytoplasm into mitochondria in an ATP- mediated reaction (1-3). Human Hsp60 contains 573 amino acids, is related to the bacteria groEL protein, and located in the mitochondria and cytoplasm, the cell surface, the extracellular space, and the peripheral blood (4-5). Under dehydration conditions, the cytoplasmic Hsp60 is quickly imported into the mitochondria by cytoplasmic Hsp70. Extracellular Hsp60 mediates apoptosis via Toll-like receptors in heart failure. Hsp60 plays a role in myelinogenesis and neurodegeneration and its defect can cause neurodegenerative pathologies. It has been suggested that Hsp60 links to diabetes, stress response, cancer and immunological disorders. Since it participates in cell signaling and key pathways, and is actively secreted by human tumor cells, Hsp60 could be used for cancer diagnosis and therapy.|
|Assay Precision||Intra-assay and inter-assay coefficients of variation were 4.8% and 7.2% respectively.|
|Synonyms||HSPD1, DKFZp459H2320, HLD4, CPN60, GROEL, HSP60, HSP65, SPG13, HuCHA60, hld4, cpn60, groel, hsp60, hsp65, spg13, chaperonin, cb863, fa04a05, fb22d10, fi27b05, sb:cb144, id:ibd2197, wu:fa04a05, wu:fb22d10, wu:fi04a12, wu:fi27b05, MIF4, MNA2, mopA, groL, crpA|
|Principle||The AssayMax Human Hsp60 ELISA (Enzyme-Linked Immunosorbent Assay) kit is designed for detection of human Hsp60 in cell culture lysates, plasma, serum, and tissue samples|
|Protocol||This assay employs a quantitative sandwich enzyme immunoassay technique that measures human Hsp60 in less than 5 hours. A polyclonal antibody specific for human Hsp60 has been pre-coated onto a 96-well microplate with removable strips. Hsp60 in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for Hsp60, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added. The color development is stopped and the intensity of the color is measured.|
|Reagent Preparation||Freshly dilute all reagents and bring all reagents to room temperature before use. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. EIA Diluent Concentrate (10x): Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8°C. Standard Curve: Reconstitute the 40 ng of Hsp60 Standard with 0.5 ml of EIA Diluent to generate a solution of 80 ng/ml. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (80 ng/ml) 1:2 with EIA Diluent to produce 40, 20, 10, 5 and 2.5 ng/ml solutions. EIA Diluent serves as the zero standard (0 ng/ml). Any remaining solution should be discarded. Fresh standard should be reconstituted the day the assay is run. Standard Point Dilution [Hsp60] (ng/ml) P1 Standard (80 ng/ml) 80.0 P2 1 part P1 + 1 part EIA Diluent 40.0 P3 1 part P2 + 1 part EIA Diluent 20.0 P4 1 part P3 + 1 part EIA Diluent 10.0 P5 1 part P4 + 1 part EIA Diluent 5.00 P6 1 part P5 + 1 part EIA Diluent 2.50 P7 EIA Diluent 0.00 Biotin Hsp60 Antibody (70x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:70 with EIA Diluent. Any remaining solution should be frozen at -20°C. Wash Buffer Concentrate (20x): Dilute the Wash Buffer Concentrate 1:20 with reagent grade water. SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1:100 with EIA Diluent. Any remaining solution should be frozen at -20°C.|
|Sample Collection||Plasma: Collect plasma using one-tenth volume of 0.1 M sodium citrate as an anticoagulant. Centrifuge samples at 2000 x g for 10 minutes and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. (EDTA or Heparin can also be used as anticoagulant.) Serum: Samples should be collected into a serum separator tube. After clot formation, centrifuge samples at 2000 x g for 10 minutes. Collect the sample and assay. Store samples at -20°C or below for up to 3 months. Avoid repeated freeze-thaw cycles. Cell Culture Lysates: Place the cell culture dish in ice and wash the cells with ice-cold PBS. Drain the PBS, then add ice-cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 0.1mM PMSF, 1g/ml leupeptin, 1g/mL 2 aprotinin, and 1g/mL pepstatin.). Scrape adherent cells off the dish and then transfer the cell suspension into a pre-cooled microfuge tube. Maintain constant agitation for 30 minutes at 4 C. Centrifuge in a microcentrifuge at 4 C. Collect fresh cell lysates. Use undiluted samples or 1:2 diluted samples with EIA Diluent and assay. The undiluted samples can be stored at -20°C or below. Tissue: Extract tissue samples with 50 mM phosphate-buffered saline (pH7.4) containing 1% Triton x-100 and centrifuge at 14000x g for 20 min. Collect the supernatant and measure the protein concentration. Use undiluted samples or 1:2 diluted samples with EIA Diluent and assay. The undiluted samples can be stored at -20°C or below.|
|Assay Procedure||Prepare all reagents, working standards and samples as instructed. Bring all reagents to room temperature before use. The assay is performed at room temperature (20 - 30 °C). Remove excess microplate strips from the plate frame and return them immediately to the foil pouch with desiccant inside. Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator. Add 50 µL of Hsp60 standard or sample per well. Cover wells with a sealing tape and incubate for two hours. Start the timer after the last sample addition. Wash five times with 200 µL of Wash Buffer manually. Invert the plate each time and decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. 3 If using a machine wash six times with 300 µL of Wash Buffer and then invert the plate, decant the contents, hit it 4-5 times on absorbent paper towel to completely remove the liquid. Add 50 µL of Biotinylated Hsp60 Antibody to each well and incubate for two hours. Wash the microplate as described above. Add 50 µL of Streptavidin-Peroxidase Conjugate to each well and incubate for 30 minutes. Turn on the microplate reader and set up the program in advance. Wash the microplate as described above. Add 50 µL of Chromogen Substrate per well and incubate for about 15 minutes or till the optimal blue color density develops. Gently tap plate to ensure thorough mixing and break the bubbles in the well with pipette tip. Add 50 µL of Stop Solution to each well. The color will change from blue to yellow. Read the absorbance on a microplate reader at a wavelength of 450 nm immediately. If wavelength correction is available, subtract readings at 570 nm from those at 450 nm to correct optical imperfections. Otherwise, read the plate at 450 nm only. Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes, which will reduce the readings.|
|Calculation of Results||Calculate the mean value of the triplicate readings for each standard and sample. To generate a Standard Curve, plot the graph using the standard concentrations on the x-axis and the corresponding mean 450 nm absorbance on the y-axis. The best-fit line can be determined by regression analysis using log-log or four-parameter logistic curve-fit. Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor. Standard Curve The curve is provided for illustration only. A standard curve should be generated each time the assay is performed.|
|Handling Advice||Prepare all reagents (working diluent buffer, wash buffer, standards, biotinylated- antibody, and SP conjugate) as instructed, prior to running the assay. Prepare all samples prior to running the assay. The dilution factors for the samples are suggested in this protocol. However, the user should determine the optimal dilution factor. Spin down the SP conjugate vial and the biotinylated-antibody vial before opening and using contents. The kit should not be used beyond the expiration date. 1|
|Material not included||Microplate reader capable of measuring absorbance at 450 nm. Pipettes (1-20 µL, 20-200 µL, 200-1000µLand multiple channel). Deionized or distilled reagent grade water|
|Storage Comment||Store components of the kit at 2-8°C or -20°C upon arrival up to the expiration date. Store SP Conjugate and Biotinylated Antibody at -20°C Store Microplate, Diluent Concentrate (10x), Wash Buffer, Stop Solution, and Chromogen Substrate at 2-8°C Opened unused microplate wells may be returned to the foil pouch with the desiccant packs. Reseal along zip-seal. May be stored for up to 1 month in a vacuum desiccator. Diluent (1x) may be stored for up to 1 month at 2-8°C. Store Standard at 2-8°C before reconstituting with Diluent. Fresh standard should be reconstituted the day the assay is run.|
|Restrictions||For Research Use only|
Cheng, Hartl, Horwich: "The mitochondrial chaperonin hsp60 is required for its own assembly." in: Nature, Vol. 348, Issue 6300, pp. 455-8, 1991 (PubMed).
Ostermann, Horwich, Neupert et al.: "Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis." in: Nature, Vol. 341, Issue 6238, pp. 125-30, 1989 (PubMed).
Reading, Hallberg, Myers: "Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor." in: Nature, Vol. 337, Issue 6208, pp. 655-9, 1989 (PubMed).
Goloubinoff, Christeller, Gatenby et al.: "Reconstitution of active dimeric ribulose bisphosphate carboxylase from an unfoleded state depends on two chaperonin proteins and Mg-ATP." in: Nature, Vol. 342, Issue 6252, pp. 884-9, 1999 (PubMed).
Itoh, Komatsuda, Ohtani et al.: "Mammalian HSP60 is quickly sorted into the mitochondria under conditions of dehydration." in: European journal of biochemistry / FEBS, Vol. 269, Issue 23, pp. 5931-8, 2002 (PubMed).
Cappello, Conway de Macario, Marasà et al.: "Hsp60 expression, new locations, functions and perspectives for cancer diagnosis and therapy." in: Cancer biology & therapy, Vol. 7, Issue 6, pp. 801-9, 2008 (PubMed).
Magen, Georgopoulos, Bross et al.: "Mitochondrial hsp60 chaperonopathy causes an autosomal-recessive neurodegenerative disorder linked to brain hypomyelination and leukodystrophy." in: American journal of human genetics, Vol. 83, Issue 1, pp. 30-42, 2008 (PubMed).
Kim, Stice, Chen et al.: "Extracellular heat shock protein 60, cardiac myocytes, and apoptosis." in: Circulation research, Vol. 105, Issue 12, pp. 1186-95, 2009 (PubMed).
Merendino, Bucchieri, Campanella et al.: "Hsp60 is actively secreted by human tumor cells." in: PLoS ONE, Vol. 5, Issue 2, pp. e9247, 2010 (PubMed).