Human Total HSP60 DuoSet IC ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
This DuoSet IC ELISA contains the basic components required for the development of sandwich ELISAs to measure total HSP60 in cell lysates. An immobilized capture antibody specific for HSP60 binds both phosphorylated and unphosphorylated HSP60. After washing away unbound material, a biotinylated detection antibody is used to detect both phosphorylated and unphosphorylated protein, utilizing a standard Streptavidin-HRP format.
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- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Available in 2, 5, and 15- (96-well) plate pack sizes
- Economical alternative to Western blot
- Capture Antibody
- Conjugated Detection Antibody
- Calibrated Immunoassay Standard or Control
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2O4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: From Costar EIA Plate (Costar Catalog # 2592) or R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
*For the Lysis Buffer, IC Diluent, and Blocking BUffer recommended for a specific DuoSet ELISA Development Kit, please see the product
Specificity of Human Total HSP60 DuoSet IC ELISA is Shown by Western Blot. Lysates prepared from Jurkat human acute T cell leukemia cells were incubated in wells coated with Human Total HSP60 Capture Antibody. Unbound material was removed by washing and bound material was solubilized in SDS gel sample buffer. The same lysate and captured proteins were electrophoresed, transferred to an Immobilon-P membrane (Millipore) and immunoblotted with Human Total HSP60 Detection Antibody. Only a single band corresponding to HSP60 was detected. To further determine specificity, recombinant human (rh) HSP27 (Catalog # 1580-HS) and rhHSP70 (Catalog # AP-100) were assayed at 400 ng/mL and did not cross-react or interfere in the assay.
Quantifications of HSP60 in Human Cell Lysates. Lysates prepared from Md from MCF-7 human breast cancer cells, HeLa human cervical epithelial carcinoma cells and Jurkat human acute T cell leukemia cells were quantified with this DuoSet IC ELISA. The same lysates were also immunoblotted (inset) with Mouse Anti-human HSP60 (Catalog # MAB1800) Antibody. The DuoSet IC ELISA results correlate well with the relative amounts of HSP60 detected by Western blot.
Preparation and Storage
The heat shock proteins are a highly conserved family of stress response proteins. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific degradative pathways. Some HSPs are expressed at low levels under normal physiological conditions but show dramatically increased expression in response to cellular stress, others are constitutively expressed. Specific HSPs play a role in regulating apoptosis by interacting directly with key components of the apoptotic pathway.
Citations for Human Total HSP60 DuoSet IC ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 3
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Cell-Free Circulating Mitochondrial DNA: A Potential Blood-Based Marker for Atrial Fibrillation
Authors: M Wiersma, DMS van Marion, EJ Bouman, J Li, D Zhang, KS Ramos, EAH Lanters, NMS de Groot, BJJM Brundel
Sample Types: Serum
Distending Pressure Did Not Activate Acute Phase or Inflammatory Responses in the Airways and Lungs of Fetal, Preterm Lambs
PLoS ONE, 2016;11(7):e0159754.
Sample Types: BALF
Sustained inflation at birth did not alter lung injury from mechanical ventilation in surfactant-treated fetal lambs.
Authors: Hillman N, Kemp M, Miura Y, Kallapur S, Jobe A
PLoS ONE, 2014;9(11):e113473.
Sample Types: BALF
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