Description

Nori Human Sirtuin 1 ELISA Kit Summary

Alternative names for Sirtuin 1: SIRT1, NAD-dependent deacetylase sirtuin-1

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Sirtuin 1, also known as NAD-dependent deacetylase sirtuin-1, is a protein that is encoded by the SIRT1 gene.^[1] Sirtuin 1 is in class I of the sirtuin family and homologs of the Sir2 gene in S. cerevisiae. SIRT1 deacetylates proteins that contribute to cellular regulation.^[2] Sirtuin 1 is downregulated in cells that have high insulin resistance and inducing its expression increases insulin sensitivity, suggesting the molecule is associated with improving insulin sensitivity.^[3] Furthermore, SIRT1 was shown to de-acetylate and affect the activity of both members of the PGC1-alpha/ERR-alpha complex, which are essential metabolic regulatory transcription factors.^[4][5][6][7] In mammals, SIRT1 has been shown to deacetylate and thereby deactivate the p53 protein. SIRT1 also stimulates autophagy by preventing acetylation of proteins (via deacetylation) required for autophagy as demonstrated in cultured cells and embryonic and neonatal tissues. This function provides a link between sirtuin expression and the cellular response to limited nutrients due to caloric restriction.^[8] SIRT1 inhibits NF-κB-regulated gene expression by deacetylating the RelA/p65 subunit of NF-κB at lysine 310.^[9] SIRT1 plays a role in activating T helper 17 cells, which contribute to autoimmune disease; efforts to activate SIRT1 therapeutically may trigger or exacerbate autoimmune disease. Lamin A is a direct activator of Sirtuin 1 during a study on progeria and resveratrol enhances the binding between Sirtuin 1 and Lamin A.^[10] Sirtuin 1 has been shown to interact with HEY2,^[11] PGC1-alpha,^[5] ERR-alpha,^[4] and AIRE.^[12]

References

1. Frye RA (1999). Biochemical and Biophysical Research Communications. 260 (1): 273–79.
2. Sinclair DA, Guarente L (2006). Scientific American. 294 (3): 48–51, 54–7.
3. Sun C, et al. (2007). Cell Metabolism. 6 (4): 307–19.
4. Wilson BJ, et al. (2010). Molecular Endocrinology. 24 (7): 1349–58.
5. Rodgers JT, et al. (2005). Nature. 434 (7029): 113–8.
6. Nemoto S, et al. (2005). The Journal of Biological Chemistry. 280(16): 16456–60.
7. Lagouge M, et al. (2006). Cell. 127 (6): 1109–22.
8. Lee IH, et al. (2008). Proc Natl Acad Sci USA. 105 (9): 3374–89.
9. Yeung F, et al. (2004). The EMBO Journal. 23 (12): 2369–80.
10. Liu B, et al. (2012). Cell Metabolism. 16 (6): 738–50.
11. Takata T, et al. (2003). Biochemical and Biophysical Research Communications. 301 (1): 250–57.
12. Chuprin A, et al. (2015). Nature Immunology. 16 (7): 737–45.

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