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Epigenetics Assay Kits

iD Sensolyte® Assay Kits

Posttranslational modifications (PTM) of histones, such as methylation, acetylation, and phosphorylation play a significant role in controlling gene expression and DNA repair. PTMs of histones have been correlated with a variety of human disease states including rheumatoid arthritis, cancer, heart disease, diabetes, neurodegenerative disorders. PTMs themselves are not sufficient to effect transcriptional activity, indicating the need for effectors proteins that bind modified residues and regulate nuclear function. These chromatin-associated proteins are gaining attention as potential therapeutic targets for epigenetic related diseases. Biotinylated histone peptides have been used to study effectors proteins translating PTMs into functional changes. 

Histone deacetylase (HDAC) enzymes modulate gene expression through the deacetylation of lysine residues on histone proteins and act as transcriptional repressors of genes. Based on their role in cell cycling, apoptosis and differentiation, HDACs have been chosen as therapeutic targets for the treatment of cancer and neurodegenerative diseases. 

Sirtuins (SIRTs) are a unique class of deacetylases (class III HDACs) that require NAD as a cofactor. Sirtuin 1 (SIRT1) is involved in regulation of tumor suppressor p53 and inhibition of apoptosis. It also represents a target for treatment of age-related diseases and type II diabetes. Sirtuin 2 (SIRT2) is a cytoplasmic protein involved in regulation of cell cycle, DNA repair and tumorogenesis. 

Histone acetyltransferases (HATs) enzymes regulate the acetylation of histones and non-histone proteins. The acetylation of the e-amino groups of lysine residues present at histone tails correlates largely with transcriptional activation, but it is also involved in DNA replication, DNA repair and protein–protein interactions. HATs play major roles in the control of cell fate and their misregulation is implicated in the development of some human tumors. HAT p300 is a transcriptional coactivator that acetylates core histones facilitating chromatin decondensation and recruiting basic RNA polymerase machinery. The p300/CBP-associated factor (pCAF) acetylates specific lysines on the N-terminal tails of histones H3 and H4.

DescriptionSize Reference USD Qty  
SensoLyte® 520 FRET Sirt1 Assay Kit *Fluorimetric* - 1 kit1 kit AS-72155 480.00Add to cart
SensoLyte® 520 FRET SIRT2 Assay Kit *Fluorimetric* - 1 kit1 kit AS-72189 480.00Add to cart
SensoLyte® 520 HDAC Activity Assay Kit *Fluorimetric* - 1 kit1 kit AS-72084 480.00Add to cart
SensoLyte® Green Sirt1 Assay Kit *Fluorimetric* - 1 kit1 kit AS-72156 480.00Add to cart
SensoLyte® Green SIRT2 Assay Kit *Fluorimetric* - 1 kit1 kit AS-72188 480.00Add to cart
SensoLyte® HAT (p300) Assay Kit *Fluorimetric* - 1 kit1 kit AS-72172 480.00Add to cart

The SensoLyte® HDAC Activity Assay Kits provide a convenient, two-step homogeneous procedure for measuring HDAC activity using a fluorogenic substrate. In the first step, an acetylated substrate is incubated with HDAC containing samples. Deacetylation of substrate sensitizes it, so that, in the second step mixing with the HDAC developer generates a fluorophore, that can be detected at Ex/Em= 354 nm/442 nm (SensoLyte® 440 HDAC Activity Assay Kit) or Ex/Em= 490 nm/520 nm (SensoLyte® 520 HDAC Activity Assay Kit). The substrate included in the kits is cell-permeable, and the assay can be used to measure HDAC activity directly in cell culture in a 96-well plate without a time-consuming cell extraction step. The SensoLyte® 520 HDAC Activity Assay Kit is highly recommended for high throughput screening of HDAC inhibitors using extracts or purified enzymes. The long wavelength fluorescence of HDAC 520 substrate is less interfered by the autofluorescence of cell components and test compounds. 

The SensoLyte® SIRT Assay Kits provide a convenient, two-step homogeneous procedure for measuring sirtuin 1 activity using a FRET (520 Kits) or fluorogenic (Green Kits) substrate. In the first step, an acetylated substrate is incubated with SIRT containing samples. Deacetylation of substrate first sensitizes it, so that, in the second step mixing with the SIRT developer generates a fluorophore, which can be detected. These kits can be used for high throughput screening of SIRT inhibitors/activators. The long wavelength fluorescence of the substrates is less interfered by the autofluorescence of cell components and test compounds. Assays are performed in a standard 96-well format. 

The SensoLyte® LSD1 Assay Kit provides a convenient procedure for screening enzyme inhibitors and for detection of purified enzyme activity. This assay consists of two steps. In the first step, LSD1 enzyme is incubated with a methylated peptide substrate and hydrogen peroxide (H2O2) is generated. In the second step, in the presence of peroxidase, H2O2 reacts with fluorogenic substrate to produce fluorescence signal, that can be measured at excitation/emission = 530 nm/590 nm. The assay can be performed in a convenient 96-well microplate format. 

The SensoLyte® HAT (p300) Assay Kit provides a convenient protocol for screening of enzyme inhibitors and for continuous assay of p300 activity. For added convenience, this kit includes two peptide substrates: histone H3 (1-21) peptide and non-histone p53 peptide (368-386). After incubation with acetyl CoA and the substrate, the p300 enzyme generates acetylated H3 or p53 peptide and CoASH. The thiol groups of CoASH can be detected with fluorogenic reagent at excitation/ emission=389nm/513nm. 

The SensoLyte® HAT (pCAF) Assay Kit provides a convenient protocol for screening of enzyme inhibitors and for continuous assay of pCAF activity. After incubation with acetyl CoA and histone H3 (1-21) peptide the pCAF enzyme generates acetylated H3 peptide and CoASH. The fluorogenic substrate reacts with thiol groups of CoASH and produces an increase of the fluorescence that can be detected at excitation/ emission=389nm/513nm. 

The SensoLyte® Histone H3 (1-21) Sampler Kit provides 8 biotinylated H3 (1-21) derived histone peptides containing various PTM residues and control non-modified H3 (1-21) biotinylated peptide. 

The SensoLyte® Methylated Histone H3 (1-21) Sampler Kit provides 9 biotinylated histone H3 (1-21) derived peptides with lysine methylated at different positions and at different degree. The kit also contains control non-modified H3 (1-21) biotinylated peptide. These peptides can be utilized to design AlphaScreen® based methyltransferase/demethylase assays and for in vitro pull-down assays.

Description



λ
Ex/Em
(nm)
Biological sample volume Lowest Detection Limit  Kit contains Assay time # of
assays in
96-well plate
HAT  Assay Kit
SensoLyte® HAT (p300)Fluorimetric 389/513
10 µl 0,78 µM • Acetyl CoA
• Histone H3 peptide (1-21)
• p53 peptide (368-386)
• HAT, recombinant p300 enzyme
• Assay buffer
• p300 inhibitor
• p300 developer
• Stop solution
• CoASH standard
1 hour 100
SensoLyte® HAT (pCAF)Fluorimetric
389/513 10 µl 0,78 µM • Acetyl CoA
• Histone H3 peptide (1-21)
• HAT, recombinant pCAF enzyme
• Assay buffer
• pCAF developer
• Stop solution
• CoASH standard
1 hour 100
HDAC Activity Assay Kit
SensoLyte® 520 HDAC Activity Fluorimetric 490/520 Example
6x104
HeLa cells
0.08 μg of HeLa protein extract/well • HDAC QXL/5-FAM substrate. The substrate included in the kit is cell-permeable, and the assay can be used to measure HDAC activity directly in cell culture in a 96-well plate without a time-consuming cell extraction step.
• Deacetylated reference standard for calibration
• HeLa nuclear extract
• Assay buffer
• Cell lysis buffer
• HDAC developer
• Trichostatin A (HDAC inhibitor)
1 hour 100
SensoLyte® 440 HDAC Activity
Fluorimetric
354/442 Example
6x104
HeLa cells
0.3 μg of
HeLa protein extract/well

• HDAC Ac-GAK (Ac)-AMC substrate. The substrate included in the kit is cell-permeable, and the assay can be used to measure HDAC activity directly in cell culture in a 96-well plate without a time-consuming cell extraction step.
• Deacetylated reference standard for calibration
• HeLa nuclear extract
• Assay buffer
• Cell lysis buffer
• HDAC developer
• Trichostatin A (HDAC inhibitor)

1 hour 100
SIRT1 Assay Kit
SensoLyte® 520 FRET SIRT1
Fluorimetric
490/520 n.d 2 ng/well • QXL/5-FAM substrate derived from a human FOXO3 sequence surrounding the deacetylation site of SIRT1. Deacetylation of the SIRT1 fluorogenic substrate by other members of sirtuin family, such as sirtuin 2 and sirtuin 3, is negligible.
• Deacetylated reference standard for calibration
• HeLa nuclear extract
• Assay buffer
• Cell lysis buffer
• HDAC developer
• Trichostatin A (HDAC inhibitor)
1 hour 100
SensoLyte® Green SIRT1
Fluorimetric
490/520 n.d n.d • QXL/5-FAM substrate derived from a human p53 sequence. Deacetylation of the SIRT1 fluorogenic substrate by other members of sirtuin family, such as sirtuin 2 and sirtuin 3, is negligible
• Deacetylated reference standard for calibration
• HeLa nuclear extract
• Assay buffer
• Cell lysis buffer
• HDAC developer
• Trichostatin A (HDAC inhibitor)
1 hour 100
SIRT2 Assay Kit
SensoLyte® 520 FRET SIRT2 Fluorimetric 490/520 50 µl 0,078 µM
• SIRT2 520 FRET substrate
• Deacetylated reference standard
• SIRT2, human recombinant
• Assay buffer
• NAD+
• Sirtuin Inhibitor
• SIRT2 Developer 10X
1 hour 100
SensoLyte® Green SIRT2 Fluorimetric
490/520 50 µl 0,156 µM • SIRT2 substrate
• Deacetylated reference substrate
• SIRT2, human recombinant
• Assay buffer
• NAD+
• Sirtuin Inhibitor
• SIRT2 Developer 10X
1 hour 100
LSD1 Assay Kit
SensoLyte® LSD1
Fluorimetric
530/590 HTP n.d • Fluorimetric substrate
• Methylated peptide as standard reference for calibration
• LSD1 enzyme
• Assay buffer
• Peroxydase enzyme
1 hour 100
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