SIRT1 Total and pSer47 Human In-Cell ELISA Kit (Fluorescent) (ab136808)
- Product nameSIRT1 Total and pSer47 Human In-Cell ELISA Kit (Fluorescent)See all SIRT1 kits ...
Intra-assay Sample n Mean SD CV% Hek293T 7.6% pSer47 6.5%
- Sample typeAdherent cells, Suspension cells
- Assay typeCell-based
- Range3000 cells/well - 50000 cells/well
- Species reactivityReacts with: Human
- Product overview
ab136808 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure the 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 monoclonal antibodies. Relative target levels are quantified using secondary antibodies conjugated to either horseradish peroxidase (HRP) or alkaline phosphatase (AP) which generate signal through the use of two spectrally distinct fluorogenic substrates. Fluorescence is measured using a standard fluorescent spectrophotometer. Optionally, antibody signal intensity can be normalized to the total cell stain Janus Green.
In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry of cultured cells. 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 fluorescence detection 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
- Storage instructionsStore at +4°C. Please refer to protocols.
Components 1 x 96 tests 1000X Anti-Mouse IgG/AP-Labeled Secondary Antibody 1 x 20µl 1000X Anti-Rabbit IgG/HRP-Labeled Secondary Antibody 1 x 20µl 100X Primary Antibody Cocktail 1 x 120µl 100X Triton X-100 1 x 1.25ml 10X Blocking Buffer 1 x 15ml 10X Phosphate Buffered Saline 1 x 100ml 10X Quenching Solution 1 x 1.5ml 400X Fluorescent Substrate Cocktail 1 x 50µl 400X Tween-20 1 x 2ml 8000X Hydrogen Peroxide 1 x 50µl Fluorescent Substrate Buffer 1 x 12ml Janus Green Stain 1 x 17ml
- Epigenetics and Nuclear Signaling
- Chromatin Modifying Enzymes
- Class III / Sir2 class
- NAD-dependent protein deacetylase sirtuin-1Regulatory protein SIR2 homolog 1SIR1_HUMANSIR2-like protein 1SIR2L1Sirt1SirtT1 75 kDa fragment
Our Abpromise guarantee covers the use of ab136808 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||In-Cell ELISA|
SIRT1 Total and pSer47 Human In-Cell ELISA Kit (Fluorescent) images
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 4°C) and secondary antibody incubation (2 hours RT) were all carried out with 1X block (ab126587) in PBS + 0.05% Tween.
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.
Specificity of Signal by In Cell ELISA. Hek293T cells were seeded on an amine coated plate at 6 k, 12 k, 25 k and 50 k and 100 k/well the day before fixation. Levels of total SIRT1 and phosphorylated protein at S47 were measured after permeabilizing with methanol at -20°C for 25 minutes. Once the methanol was washed with PBS, treatment with 1:100 dilution of Lambda Phosphatase 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.
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 pS47 (BOTTOM) are shown after background subtraction.
References for SIRT1 Total and pSer47 Human In-Cell ELISA Kit (Fluorescent) (ab136808)
ab136808 has not yet been referenced specifically in any publications.