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Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit (Colorimetric)

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For measuring enzymatic activity or inhibition levels of TET

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Suggested Workflow
Nuclear Protein Extraction
 
 
Demethylase Assay
 
Schematic procedure for the Epigenase™ 5mC-Hydroxylase TET Activity/Inhibition Assay Kit (Colorimetric).
Demonstration of high sensitivity and specificity of the TET1 activity/inhibition assay achieved by using recombinant TET1 with the Epigenase 5-mC Hydroxylase TET Activity/Inhibition Assay Kit (Colorimetric).
Demonstration of high sensitivity and specificity of the TET activity assay achieved by using nuclear extracts with the the Epigenase 5-mC Hydroxylase TET Activity/Inhibition Assay Kit (Colorimeric). Nuclear extracts were prepared from mouse ES cells by using the EpiQuik Nuclear Extraction Kit (Cat. No. OP-0002).
Illustrated standard curve generated with the kit's TET assay standard.
Input Type: Nuclear Extracts, Purified Enzyme
Research Area: DNA Methylation
Target Application: Activity Measurement
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-3086-4848 assays $317.00 
P-3086-9696 assays $539.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The Epigenase™ 5mC-Hydroxylase TET Activity/Inhibition Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents for measuring activity/inhibition of total cytosine oxygenase TET enzymes in nuclear extracts or purified TET isoforms (TETs 1-3) from a broad range of species such as mammals, plants, fungi, and bacteria, and in a variety of forms including, but not limited to cultured cells and fresh and frozen tissues.  The kit has the following advantages and features:

  • 5 hour colorimetric assay procedure with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 5 hours.
  • Directly measures TET hydroxylase activity via a straightforward detection of TET-converted hydroxymethylated products.
  • Innovative kit composition enables background signals to be extremely low and allows the assay to be simple, accurate, reliable, and consistent.
  • Either cell/tissue extracts or purified TET proteins can be used, which allows for detection of inhibitory effects of TET hydroxylase inhibitors in vivo and in vitro.
  • Novel assay principle allows high sensitivity to be achieved. The activity can be detected from as low as 20 ng of purified TET1 hydroxylase.
  • A hydroxymethylated standard is included, which allows the specific activity of TET hydroxylases to be quantified.  
  • Strip microplate format makes the assay flexible for manual or high throughput analysis (96 assays).

Background Information
5-hydroxymethylcytosine, as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in the human and mouse brain and embryonic stem (ES) cells. In mammals, it can be generated by oxidation of 5-methylcytosine, a reaction mediated by the ten-eleven translocation (TET) family of cytosine oxygenases. 

The TET family of cytosine oxygenases includes TET1, TET2, and TET3. These TET proteins may promote DNA demethylation by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC and further to 5caC (5-carboxylcytosine) through hydroxylase activity. It was shown that genomic 5hmC levels correlate with TET hydroxylase activity. In addition,  TET1 was shown to have dual functions in transcription activation and repression by binding different target genes in ES cells. TET1 is also a fusion partner of the MLL gene in acute myeloid leukemia and is considered an oncoprotein. TET2 is found to be frequently mutated in leukemia and considered to act as tumor suppressor. TET3 was demonstrated to play a unique role in DNA methylation reprogramming processes in the mammalian zygote. Thus, activating tumor suppressor TET enzymes such as TET2 or inhibiting oncoprotein TET enzymes such as TET1 would be important in benefiting cancer diagnositcs and developing new target-based cancer therapeutics.

Principle & Procedure
In this assay, a methylated substrate is stably coated onto the microplate wells. Active TETs (not included) bind to the substrate and convert the methylated substrate to hydroxymethylated products. The TET-converted hydroxymethylated products can be recognized with a specific antibody. The ratio or amount of hydroxymethylated products, which is proportional to enzyme activity, can then be colorimetrically measured by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The activity of the TET enzyme is in turn proportional to the optical density intensity measured.

Product Components

WB (10X Wash Buffer)
TAB (TET Assay Buffer)
TS (10 X TET Substrate)*
BS (Binding Solution)
HC5 (TET Assay Standard, 20 µg/ml)*
HC6 (Capture Antibody, 1000 µg/ml*
HC7 (Detection Antibody, 400 µg/ml)*
HC8 (Enhancer Solution)*
DS (Developer Solution)
SS (Stop Solution)
Co-factor 1*
Co-factor 2*
Co-factor 3*
8-Well Assay Strips (With Frame)

* For maximum recovery of the products, centrifuge the original vial prior to opening the cap.

 

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations

Shenoy N et. al. (July 2017). Upregulation of TET activity with ascorbic acid induces epigenetic modulation of lymphoma cells. Blood Cancer J. 7(7):e587.

Prasad R et. al. (May 2017). Honokiol inhibits ultraviolet radiation-induced immunosuppression through inhibition of ultraviolet-induced inflammation and DNA hypermethylation in mouse skin. Sci Rep. 7(1):1657.

Röhr D et. al. (April 2017). Sodium-dependent Vitamin C transporter 2 deficiency impairs myelination and remyelination after injury: Roles of collagen and demethylation. Glia.

Kowluru RA et. al. (January 2017). Role of oxidative stress in epigenetic modification of MMP-9 promoter in the development of diabetic retinopathy. Graefes Arch Clin Exp Ophthalmol.

Ishikawa K et. al. (February 2015). Long-term pancreatic beta cell exposure to high levels of glucose but not palmitate induces DNA methylation within the insulin gene promoter and represses transcriptional activity. PLoS One. 10(2):e0115350.

Niu Y et. al. (February 2015). Oxidative stress alters global histone modification and DNA methylation. Free Radic Biol Med.

He XB et. al. (December 2014). Vitamin C Facilitates Dopamine Neuron Differentiation in Fetal Midbrain Through Tet1- aND Jmjd3-Dependent Epigenetic Control Manner. Stem Cells.

Dubois-Chevalier J et. al. (September 2014). A dynamic CTCF chromatin binding landscape promotes DNA hydroxymethylation and transcriptional induction of adipocyte differentiation. Nucleic Acids Res.

Scola G et. al. (April 2014). Lithium reduces the effects of rotenone-induced complex I dysfunction on DNA methylation and hydroxymethylation in rat cortical primary neurons. Psychopharmacology (Berl).

Nettersheim D et. al. (December 2013). Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development. PLoS One. 8(12):e82881.

Coulter JB et. al. (October 2013). Hydroquinone increases 5-hydroxymethylcytosine formation through ten eleven translocation 1 (TET1) 5-methylcytosine dioxygenase. J Biol Chem. 288(40):28792-800.

Haseeb et. al. (November 2012). IL-1 And TNF- Regulate The Global And Locus-Specific Hydroxymethylation Of Genomic DNA By Modulating The Expression And Activity Of Tet-1 In Human OA Chondrocytes. Arthritis Rheum. Abstract

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