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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.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
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.
|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|
Our Abpromise guarantee covers the use of ab136807 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||Use at an assay dependent concentration.|
ab136807 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"