The Epigenase™ Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay Kit (Colorimetric) is a complete set of optimized buffers and reagents for measuring the activity/inhibition of total TDG enzyme in nuclear extracts or purified TDG enzymes from a broad range of species such as mammals, plants, fungi, and bacteria, 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:
- Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 5 hours.
- Directly measures TDG activity without the need for DNA cleavage, electrophoresis, or chromatography.
- Both cell/tissue extracts and purified TDG proteins can be used, which allows for detection of inhibitory effects of TDG 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 TDG protein.
- TDG assay standard is conveniently included, which allows for specific quantification of TDG activity.
- Strip microplate format makes the assay flexible: manual or high throughput analysis (96 assays).
DNA demethylation has been demonstrated to occur through at least two different pathways: (1) active deamination of 5-mC to T by an AID/APOBEC enzyme, giving a G/T mismatch that is converted to G/C by thymine DNA glycosylase (TDG) and then subsequently base excision repair (BER); and (2) iterative oxidation of 5-mC by TET enzyme to 5-hmC and further to 5-formycytosine (5-fC) and 5-carboxylcytosine (5-caC). Both 5-fC and 5-caC can be rapidly excised by TDG to allow subsequent BER processing which results in 5-fC and 5-caC converting back to unmodified cytosine.
TDG belongs to the TDG/mug DNA glycosylase family. Besides playing a critical role in active DNA methylation, TDG also removes thymine moieties from G/T mismatches by hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of DNA and the mismatched thymine. It has been shown that TDG participates in the regulation of embryonic and germ cell development, mediates cellular defense against genetic and epigenetic mutations, and mediates DNA-directed cytotoxicity. In addition, TDG is a proposed tumor suppressor candidate. Thus, the measurement of TDG activity would be important as TDG may be involved in preventing tumorigenesis and determining cellular response to chemotherapy through DNA demethylation and BER, thereby benefiting cancer diagnostics and new target-based cancer therapeutics.
The currently used methods for TDG activity detection involve DNA cleavage followed by electrophoresis, which can be time consuming and inconvenient, with low throughput and high cost. To address this issue, Epigentek developed and offers the Epigenase™ Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay Kit (Colorimetric).
Principle & Procedure
In this assay, a 5-fC DNA substrate is stably coated onto microplate wells. Active TDG binds to the substrate and excises 5-fC from the substrate by a nicking reaction. The remaining un-excised 5-fC in the substrate will be recognized with a high affinity 5-fC DNA antibody. The ratio or amount of 5-fC in the substrate, which is inversely 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 TDG enzyme is in turn inversely proportional to the optical density intensity measured.
Starting Materials & Input Amount
The amount of nuclear extracts for each assay can be 2 µg to 20 µg with an optimal range of 5-6 µg. The amount of purified enzymes can be 20 ng to 1 µg, depending on the purity and catalytic activity of the enzymes.
Fig. 1. Schematic procedure of the Epigenase™ Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay (Colorimetric).
Fig. 2. Illustrated standard curve generated with the TDG assay standard from the Epigenase™ Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay (Colorimetric).
Fig. 3. Demonstration of high sensitivity and specificity of the TDG activity assay achieved by using nuclear extracts with the the Epigenase ™ Thymine DNA Glycosylase (TDG) Activity/Inhibition Assay Kit (Colorimetric). Hela Nuclear extracts were used in the assay.