SIRT1 Total and pSer47 Human In-Cell ELISA Kit (Colorimetric) (ab136807)

Overview

• Product nameSIRT1 Total and pSer47 Human In-Cell ELISA Kit (Colorimetric)See all SIRT1 kits ...
• Precision

  Intra-assay

  Sample	n	Mean	SD	CV%
  Hek293T				5.9%
  pSer47				5.5%
• Sample type
  Adherent cells, Suspension cells
• Assay typeCell-based
• Range
  6000 cells/well - 100000 cells/well
• Species reactivity
  Reacts with: Human
  
• Product overview

  ab136807 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure levels of SIRT1 total protein and phosphorylated at Ser47 in cultured cells. Cells are fixed in a microplate and targets of interest are detected with highly specific, well-characterized antibodies. Relative target levels are quantified using a HRP labeled secondary antibodies and a standard spectrophotometer capable kinetic reading at 600nm or endpoint reading at 450nm. Optionally, antibody signal intensity can be normalized to the total cell stain Janus Green. The pSer47 rabbit primary antibody could also be replaced by a different previously optimized rabbit antibody of the customer’s choice.

• 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 proteins such as Histone H3, PGC1a, FOXO1, FOXO3, p53, Notch, NF-kB, HIF1a, LXR, FXR, SREBP1c, 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 Ser27, Ser47 and Thr530 resulting in deacetylation of histone H3 but not of p53. On the other hand DYRK1 and DYRK3 phosphorylate SIRT1 at residue T22 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.

  
  In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry of cultured cells with a Horseradish peroxidase detector antibody. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of the method and the ability to run up to 96 samples in parallel. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins and their post-translational modifications, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts. Finally, the signal can be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.

• Tested applicationsIn-Cell ELISA more details

Properties

• Storage instructionsStore at +4°C. Please refer to protocols.

• Components	1 x 96 tests
  100X GAM HRP Label	1 x 125µl
  100X GAR HRP Label	1 x 125µl
  100X Mouse Anti-SIRT1 Primary Antibody	1 x 120µl
  100X Rabbit Anti-SIRT1 pSer47 Primary Antibody	1 x 120µl
  100X Triton X-100	1 x 1.25ml
  10X Blocking Buffer	1 x 15ml
  10X Phosphate Buffered Saline	1 x 100ml
  1X HRP Development Solution	1 x 24ml
  400X Tween-20	1 x 2ml
  Janus Green Stain	1 x 17ml

• Research Areas
  • Metabolism
  • Types of disease
  • Obesity

  • Kits/ Lysates/ Other
  • Kits
  • ELISA Kits
  • In Cell ELISA Kits
  • Phosphoprotein ICE kits

  • Microbiology
  • Interspecies Interaction
  • Host Virus Interaction

  • Tags & Cell Markers
  • Subcellular Markers
  • Nucleus
  • Other Nuclear Bodies

  • Epigenetics and Nuclear Signaling
  • Chromatin Modifying Enzymes
  • Acetylation
  • HDACs
  • Class III / Sir2 class

  • Cell Biology
  • Apoptosis
  • Intracellular
  • p53 Pathway

Applications

SIRT1 Total and pSer47 Human In-Cell ELISA Kit (Colorimetric) images

• 

  Figure 1. Dynamic range of Hek293T cells.
  Figure 1. Dynamic range of Hek293T cells. Cells were seeded the day before at the specified cell densities. The signal was obtained using this kit as described. Total SIRT1 (TOP) and SIRT1 phospho S47 (BOTTOM) are shown after background subtraction.
• 

  Figure 2. Specificity of Signal by In Cell ELISA.
  Figure 2. Specificity of Signal by In Cell ELISA. Hek293T cells were seeded on an amine coated plate at 12 k, 25 k and 50 k/well the day before fixation. Levels of total SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with methanol at -20ᵒ C for 25 minutes. Once the methanol was washed with PBS, treatment with 1:100 dilution of LP was carried out at 40˚C for 45 minutes on a plate heater. Blocking and antibody incubations were carried out according to this protocol. Data is shown as the mean of different wells at different cell densities after normalization with Janus green.
• 

  Figure 3. Specificity of Signal by Immunocytochemistry.
  Figure 3. Specificity of Signal by Immunocytochemistry. Hek293T cells were seeded on glass coverslips and allowed to adhere overnight. Levels of SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with 0.1% Triton (Panel 1) and methanol (Panel 2) followed by Lambda phosphatase treatment (Panel B). The total SIRT1 signal was labeled with GAM-488 and the SIRT1 pS47 with GAR-594. Panel B shows a reduction in phosphorylation due to LP treatment.
• 

  Figure 4. Specificity of signal by Western Blot.
  Figure 4. Specificity of signal by Western Blot. Western Blot was run on a 4-15% gradient acrylamide gel. Samples were loaded as follows from left to right: (1) 40 µg of control Hek293T cell extract, (2) 40 µg of mock treated Hek293T cell extract, (3) 40 µg of 1:100 LP treated Hek293T cell extract, (4) 40 µg of 50 nM calyculin treated Hek293T cell extract and (5) 40 µg of 0.5% DMSO treated Hek293T cell extract. Membrane blocking (1 hour RT), primary antibody incubation (overnight 4ᵒC) and secondary antibody incubation (2 hours RT) were all carried out with 1X block (ab126587) in PBS + 0.05% Tween.

Protocols

Protocol Booklet