SIRT1 Total and pSer47 Human In-Cell ELISA Kit (IR) (ab135394)

Overview

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

  Intra-assay

  Sample	n	Mean	SD	CV%
  Total SIRT1				4.3%
  pSer47				5.3%
• Sample type
  Adherent cells, Suspension cells
• Assay typeCell-based
• Sensitivity
  > 3000 cells/well
• Range
  3000 cells/well - 50000 cells/well
• Species reactivity
  Reacts with: Human
  
• Product overview

  ab135394 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 an IRDye®-labeled Secondary Antibody Cocktail and IR imaging system. Optionally, antibody signal intensity can be normalized to the total cell stain Janus Green.

• 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.

• Tested applicationsIn-Cell ELISA more details

Properties

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

• Components	1 x 96 tests
  1000X IRDye® Labeled Secondary Antibody Cocktail	1 x 24µl
  100X SIRT1 Total and pSer47 Primary Antibody Cocktail	1 x 120µl
  100X Triton X-100	1 x 500µl
  10X Blocking Buffer	1 x 10ml
  10X Phosphate Buffered Saline	1 x 100ml
  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 (IR) images

• 

  Specificity of Signal by Immunocytochemistry/ Immunofluorescence
  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.
• 

  Specificity of Signal by In Cell ELISA
  Hek293T cells were seeded on an amine coated plate at 12k, 25k and 50k/well the day before fixation. Levels of total SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with methanol at -20⁰C 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.
• 

  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.

Protocols

Protocol Booklet