AnaSpec, EGT Group is pleased
to offer a wide selection of highly sensitive HDAC (HDAC, SIRT1 &
SIRT2) and HAT (CAF & p300) SensoLyte® assay kits. These assays are fluorimetric,
some of which are FRET (fluorescence resonance energy transfer)-based.
Histones are the chief protein components of chromatin. They act as spools
around which DNA winds. Covalent modification of histone proteins through acetylation
and deacetylation affects chromatin structure and regulates gene expression.
Histone hyperacetylation is well correlated with increased transcription, whereas
hypoacetylation correlates with transcriptional repression.1 Histone
deacetylases (HDACs), which catalyze the removal of acetyl groups from a ε-N-acetyl
lysine amino acid on a histone, act as transcriptional repressors of genes.
Histone deacetylases have been grouped into three classes. Class I (HDAC 1,
2, 3, 8) and Class II (HDAC4, 5, 6, 7, and 9) are zinc-containing hydrolase
enzymes.2,3 The third class of deacetylases consists of the members
of the sirtuin family of enzymes (Sir 1 to 7).4 Inhibitors of HDAC
classes I and II are being studied as a treatment for cancer and neurodegenerative
diseases such as Huntington's and Alzheimer's diseases.5-7 The Sirtuin
1 (class III) enzyme represents a target for treatment of age-related diseases
and type II diabetes.8,9
Histone acetyltransferases (HATs) regulate
the acetylation of histones and non-histone proteins.10, 11 The acetylation of the ε-amino groups of lysine
residues present at histone tails correlates largely with transcriptional activation,
and is also involved in DNA replication, DNA repair and protein–protein interactions.12
Figure 1. HDAC substrates were incubated with HeLa nuclear extracts, followed
by incubation of a Trichostatin A-containing developer. The substrate in SensoLyte®
520 provides higher sensitivity and better linear range.
Figure 2. The SensoLyte® Green and 520 FRET SIRT1 substrates provide
better signal/background ratio than the AMC-containing substrate.
Figure 3. AMC, Green and FRET SIRT2 substrates were incubated with sirtuin 2
enzyme. The substrates in the SensoLyte® 520 FRET and Green SIRT
assay kits provide superior signal/background ratio.
Figure 4. Inhibitor studies. The calculated IC50 for sirtuin
inhibitor Ro-31-8220 was about 2 mM for both enzymes in the fluorogenic and
FRET assays. SIRT2 was also tested with suramin. The calculated IC50 were
8.7 mM and 19.7 mM for fluorogenic and FRET substrates, respectively.
Other Assay Kits
Histone H3 (1 - 21) Sampler Kit *Biotinylated*
Methylated Histone H3 (1 - 21) Sampler Kit *Biotinylated*
H2B (acetyl K5), polyclonal
& H2A (paired1)
View the following posters that had been presented at
Development of Histone Deacetylase Activity Assay Using a Novel Long Wavelength Fluorogenic Substrate
Design of Novel Fluorogenic and FRET Substrates for the Detection of Sirtuin Activity
1. Sterner, DE. et al. Microbiol. Mol. Biol. Rev. 64, 435 (2000).
2. De Ruijter, AJ. et al. Biochem. J. 370, 737 (2003).
3. Verdin, E. et al. Trends Genet. 19, 286 (2003).
4. Blander, G. and L. Guarente Annu. Rev. Biochem. 73, 417 (2004).
5. Huang, L. J. Cell Physiol. 209, 611 (2006).
6. Dompierre, JP. et al. J. Neurosci. 13, 3571 (2007).
7. Langley, B. Curr. Drug Targets CNS Neurol. Disord. 4, 41 (2005).
8. Trapp, J. and Jung, M. Curr. Drug Targets 7, 1553 (2006).
9. Porcu, M., and Chiarugi, A. Trends Pharmacol Sci. 26, 94 (2005).
10. Roth, SY. et al. Annu Rev Biochem. 70, 81 (2001).
11. Glozak, MA. et al. Gene. 363, 15 (2005).
12. Turner, BM. Bioessays 22, 836 (2000).