| Field | Specification |
|---|---|
| Mfr No | |
| Alternative Names | SIR2L3; mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase|silent mating type information regulation 2; S.cerevisiae; homolog 3|sir2-like 3|sirtuin 3|sirtuin type 3 |
| Product Type | |
| Sensitivity | |
| UniProt # |
Scientific Background
SIRT3 is a soluble protein located in the mitochondrial matrix, and contains a mitochondrial processing peptide at the N-terminus. A set of crystal structures of human SIRT3 have been solved, including an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide and a structure with the dethioacetylated peptide bound. These structures show the conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptid.
Assay Principle
This assay employs a two-site sandwich ELISA to quantitate SIRT3 in samples. An antibody specific for SIRT3 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and anySIRT3 present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for SIRT3 is added to the wells. After washing, Streptavidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of SIRT3 bound in the initial step. The color development is stopped and the intensity of the color is measured.
Performance Specifications
| Sensitivity | 0.112 ng/mL |
|---|---|
| Detection Range | 0.312-20 ng/mL |
| Total Assay Time | 3.5–5 hours |
| Compatible Sample Types | serum, plasma, cell culture supernatant, tissue homogenate |
| Species Reactivity | Human |
| Detection Method | Colorimetric (TMB/HRP) |
| Storage | 4°C (short-term); -20°C (long-term) |
✓ Research-Grade Validation
Safety & Regulatory
Handle reagents in accordance with institutional biosafety guidelines. Refer to the Safety Data Sheet (SDS) for complete hazard and handling information. Contains components that may require special disposal procedures per local regulations.
This kit is validated for use with serum, plasma, cell culture supernatant, tissue homogenate. For unlisted matrices (e.g., tissue lysate, urine), perform a spike-and-recovery experiment to confirm assay performance before generating reportable data. Sample dilution in the kit's provided diluent is recommended to minimize matrix interference.
The minimum detectable concentration (sensitivity) of this kit is 0.112 ng/mL. Values below this threshold should be reported as below the limit of detection (<LOD) and should not be extrapolated from the standard curve.
The total assay time from sample addition to absorbance reading is approximately 3.5–5 hours, including all incubation, wash, and substrate steps. Hands-on time is typically 1–2 hours; most steps involve passive plate incubation. Plan the assay as a single uninterrupted session for best results.
Standard components of this Sandwich ELISA Kit typically include: pre-coated microplate (96-well strip format), lyophilized or liquid recombinant SIRT3 standard, detection antibody, streptavidin-HRP conjugate, TMB substrate, stop solution, wash buffer concentrate, and sample/standard diluent. Refer to the kit insert or datasheet for the exact component list and storage requirements.
This kit uses colorimetric (TMB/HRP) detection and requires a standard microplate absorbance reader capable of measuring at 450 nm. A reference wavelength of 570 nm or 630 nm is recommended to reduce background. No specialized fluorescence or luminescence reader is needed. Ensure the instrument is calibrated and the plate is clean and free of condensation before reading.
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