(MS1104)
SIRT1 pSer47 Human ELISA Kit (ab135392)
Overview
- Product nameSIRT1 pSer47 Human ELISA KitSee all SIRT1 kits ...
- Precision
Intra-assay Sample n Mean SD CV% Overall 10 5.4% - Sample typeCell culture extracts, Tissue Extracts, Cell Lysate
- Assay typeQuantitative
- Sensitivity2 µg/ml
- Range2 µg/ml - 1000 µg/ml
- Recovery
Sample specific recovery Sample type Average % Range Serum 75 63% - 91% Cell culture media 74 61% - 87% Extraction Buffer = 94 88% - 106% - Species reactivityReacts with: Human
Does not react with
Mouse, Rat - Product overview
This SIRT1 phospho S47 ELISA Kit is an enzyme immunoassay developed for the sensitive detection of phosphorylated SIRT1 at residue S47. The kit has a detection sensitivity limit in Hek293T cells of 2 µg/mL of lysate. Each kit provides sufficient reagents to perform up to 96 assays including standard curve with a reference sample and test samples.
The assay employs a SIRT1 specific antibody coated onto well plate strips. Samples are pipetted into the wells and SIRT1 protein present in the sample is bound to the wells by the immobilized antibody. The wells are washed and a primary anti-phospho S47 detector antibody is added. After washing away unbound detector antibody, HRP-conjugated secondary detector antibody (HRP Label) specific for the primary detector antibody is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of phosphorylated SIRT1 at serine47 bound. The developing blue color is measured at 600 nm. Optionally the reaction can be stopped by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm.
- Notes
SIRT1 - silent mating type information regulation 2 homolog - (homolog of yeast Sir2) is a member of the sirtuins family of deacetylases. Sirtuin1 deactylates a growing number of protein such as Histone H3, PGC1a, FOXO1, FOXO3, p53, Notch, NF-kB, HIF1a, LXR, FXR, SREBP1c and therefore affecting a wide array of processes such as epigenetic silencing, apoptosis, senescence, adipogenesis, fatty acid oxidation, insulin secretion, glycolysis, gluconeogenesis and muscle differentiation. Furthermore SIRT1 may serve as a cytosolic NAD+/NADH sensor and may also regulate the circadian clock of the cell in response to metabolic conditions.
The activity of SIRT1 is regulated by gene expression, post-translational modification (phosphorylation and sumoylation), complex formation, substrate availability (NAD+/NADH, NAD+ precursors such as nicotinamide) and plant polyphenols such as resveratrol. Activation of SIRT1 by phosphorylation is carried out by the cyclin B-CDK1 complex, the JUN N-terminal kinase (JNK) and by DYRK1 and DYRK3. Cyclin B-CDK1 phosphorylates SIRT1 at residues thr530 and s540 which in turn affects progression through the cell cycle. JNK phosphorylates SIRT1 at residues S27, S47 and Thr530 resulting in deacetylation of histone H3 but not of p53. On the other hand DYRK1 and DYRK3 phosphorylate SIRT1 at residue Thr522 leading to deacetylation of p53 and prevention of apoptosis within the context of genotoxic stress.
Pharmacological activation of sirtuins is thought to be beneficial not only for diseases relating to metabolism, such as type 2 diabetes and obesity, but also for neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.
- Tested applicationsSandwich ELISA more details
Properties
- Storage instructionsStore at +4°C. Please refer to protocols.
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Components 1 x 96 tests 10X Blocking Buffer 1 x 6ml 10X HRP Label 1 x 1ml 10X SIRT1 pSer47 Detector Antibody 1 x 1ml 20X Buffer 1 x 20ml Extraction Buffer 1 x 15ml SIRT1 Microplate 1 unit TMB Development Solution 1 x 12ml - Research Areas
Contains 1 deacetylase sirtuin-type domain.
modificationsS-nitrosylated by GAPDH, leading to inhibit the NAD-dependent protein deacetylase activity.
Target information above from: UniProt accession
Q96EB6
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010)
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Alternative names
- hSIR2hSIRT1NAD-dependent deacetylase sirtuin-1
- Regulatory protein SIR2 homolog 1SIR2-like protein 1SIR2L1Sirt1SIRT1_HUMAN
see all
- Entrez Gene: 23411 Human
- SwissProt: Q96EB6 Human
- Unigene: 369779 Human
Applications
Our Abpromise guarantee covers the use of ab135392 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Notes |
|---|---|
| Sandwich ELISA | sELISA |
SIRT1 pSer47 Human ELISA Kit images
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Typical Standard CurveExample of positive control sample standard curve. A dilution series of extract in 1X Incubation Buffer in the working range of the assay. The extract was prepared with Hek293T cells grown in High Glucose DMEM supplemented with 10% FCS -
The SIRT1 phospho S47 ELISA specifically measures the phosphorylated serine.Hek293T extracts were left untreated (control), treated with heat only at 34˚C (Mock) or treated with 1:100 dilution of λ Ppase at 34˚C. Samples were loaded at 750 µg/mL on the plate and measured following the kit’s protocol. Treatment of Hek293T extracts with λ Ppase decreases the signal to background levels. -
Western BlotThe detector antibody used in this kit specifically detects the phosphorylated SIRT1 as determined by Western blotting. Hek293T extracts were left untreated (lane 1), treated with heat only (lane 2) or treated with 1:100 dilution of λ Ppase at 34˚C (lane 3). Samples were then diluted in SDS-PAGE buffer and loaded at 30 µg/well. Membranes were blocked with the kit’s blocking reagent at 1X concentration and incubated with either the detector antibody against SIRT1 phospho S47 (A) or the capture antibody against total SIRT1 (B) and label with secondary antibodies conjugated to HRP. λ Ppase completely dephosphorylates SIRT1 without affecting the protein levels.
Protocols
References for SIRT1 pSer47 Human ELISA Kit (ab135392)
ab135392 has not yet been referenced specifically in any publications.
