Product Details
| Cat # +Size | K4923-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | This ELISA kit is used for the in vitro quantitative determination of human Sirtuin 1 in the range of 0.031 – 2 ng Sirtuin 1/ml and a detection limit of 30 pg/ml (based on adding two standard deviations to the mean value of the (50) zero standards). |
| Features & Benefits | • Simple procedure • Fast and convenient • High-throughput adaptable • Cross Reactivity: No significant cross-reactivity or interference between this analyte and its analogues was observed. |
| Kit Components | • Pre-coated Microtiter Plate • Wash Buffer (10X) • Diluent (5X) • Lysis Buffer (10X) • Detection Antibody • Detector 100X (Hrp conjugated anti-IgG) • Human NQO1 Standard (lyophilized, 40 ng) • Human NQO1 QC Sample (lyophilized) • TMB Substrate Solution • Stop Solution • Plate Sealers |
| Storage Conditions | 4°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Sirtuin 1 is the human ortholog for the yeast Sir2 (silent information regulator 2) protein regulating epigenetic gene silencing as a possible antiaging effect. Sirtuin 1 is an NAD(+)-dependent histone deacetylase, which deacetylate lysines 9 and 14 of histone H3 and lysine-16 of histone H4, involved in various cellular functions such as transcription, energy sensing, and differentiation. Sirtuin1 plays an important role in regulating adipogenesis via repression of PPAR and the gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator PGC1A deacetylated at specific lysine residues in an NAD(+)-dependent manner. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human Sirtuin 1 in cells. A monoclonal antibody specific for Sirtuin 1 has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, Sirtuin 1 is recognized by the addition of a purified polyclonal antibody specific for Sirtuin 1 (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of Sirtuin 1 in the samples. This ELISA is specific for the measurement of natural and recombinant human Sirtuin 1. It does not cross-react with human Sirtuin 2, human Sirtuin 5, human Sirtuin 6, human adiponectin, human resistin, human RBP4, human vaspin, human progranulin, human GPX3, human FTO, human Nampt, human leptin, mouse FTO, mouse Nampt. The assay range is 0.031 – 2 ng Sirtuin 1/ml and a detection limit of 30 pg/ml (based on adding two standard deviations to the mean value of the (50) zero standards).
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What is a sandwich ELISA?
The analyte of interest in a sample is "sandwiched" by an immobilized capture antibody coated on the plate and a labeled antibody used for detection. Sandwich ELISA is very sensitive. The density of color is proportional to the amount of analyte captured from the samples and can be quantified when compared with standard curve.
What is a competitive ELISA?
The endogenous unlabled analyte of interest in a sample is "competed" by the exogenous labeled antigen coated on the plate for a limited amount of antibody binding sites. Therefore, the lower signal indicate higher concentration of the analyte.
What dilution range should I use for the samples?
A preliminary experiment is always recommended when working with new samples and ELISA Kits to ensure all results fall within the detection range.
Can I extend the standard curve (in either direction)?
Extended standard curve is not recommended by BioVision.
What type of software is needed to graph a 4-parameter or 5-parameter curve?
SoftMax Pro by Molecular Devices, SigmaPlot® by Systat Software Inc., or others can be used for this purpose.
Low absorbance; No signal
Target present below detection limits of assay
Reduce dilution factor to increase sample concentration (Preliminary experiment is recommended for optimal dilution range)
Incompatible sample types
Use samples recommended by the kit
Standard dilutions are unstable
Use fresh standard dilutions
Buffers/substrates are contaminated
Use new buffers and substrates
Insufficient incubation time
Extend incubation time
Cover or seal plates during all incubation
Plate washings too vigorous
Pipette wash buffer gently or make sure the automatic wash system has correct pressure
Wells scratched with pipette or washing tips
Use caution when dispensing and aspirating into and out of wells
Incubation temperature too low
Ensure incubations are carried out at temperatures recommended by the manual
Reagents are cold
Bring all reagents to room temperature before each assay
Plate read at incorrect wavelength
Ensure the plate reader is set at the correct wavelength recommended on the protocol
High Background
Insufficient washing
Increase number of washes; Add a 30 second soaking step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Residual buffer/substrates remain in wells
Remove all residual buffer and substrates at the end of each wash
Too much HRP-Streptavidin
Centrifuge and mix the substrate vial before use to avoid percipitation and inconsistent HRP concentration
Excessive incubation time
Reduce incubation time
Substrate incubation carry out in light or wait too long to read plate after adding stop solution
Avoid light during incubation and read signals immediately after adding stop solution
Standards improperly reconstituted or diluted
Briefly spin vials before opening; inspect for undissolved material after reconstitution
Poor standard curve
Standard improperly diluted
Confirm dilutions are made correctly
Standard dilutions are unstable
Use freshly diluted standards
Pipetting error
use calibrated pipette and stay consistent between each pipette
Curve doesn't fit scale
Try plotting using different scales (log-log, 4 parameter logistic, 5 parameter etc.)
Large intra-coefficient of variation (CV)
Bubbles in wells
Avoid bubbles during experiment, eliminate all bubbles prior to reading
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Wells not washed equally/thoroughly
Use calibrated (automated) multi-channel pipette for consistent handling, make sure all parts of the automated washer are unobstructed
Edge effects
Seal the plate completely during incubation
Incomplete reagent mixing
Make sure all reagents are mixed thoroughly during each step
Inconsistent sample preparation or storage
Consistent sample preparation. Optimize sample storage conditions and minimize freeze and thaw cycles
Stacked plates
Avoid stacking plates during incubation
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
Large inter-coefficient of variation (CV))
Insufficient washing
Increase number of washes; add a 30 second soak step in between washes; at the end of each washing step, invert plate on absorbent tissue to completely drain all wells
Inconsistent incubation temperature
Make sure to follow recommended incubation temperatures. Avoid fluctuations in temperature due to environmental conditions.
Plate sealers not used or reused
Cover assay plate with plate sealer, use a fresh sealer each time to prevent contamination
Kondo, Aki et al. (2016) Silent information regulator T1 in aqueous humor
of patients with cataract, Clin Ophthalmol. 2016 Feb 15;10:307-12.
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