TET2
- Demethylase
- Oxidizes 5-methylcytosine (5mC) through an iterative reaction into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxycytosine (5caC)
- Enables enzymatic strategies for sensitive detection of epigenetic modifications to cytosines
| Catalog # | Concentration | Size |
|---|---|---|
| M1524S | 8,000 units/ml | 400 units |
| M1524L | 8,000 units/ml | 2,000 units |
Product Information
TET2 (Tet methylcytosine dioxygenase 2) is a demethylase and ten-eleven translocation protein.
TET enzymes play a crucial role in DNA demethylation by oxidizing 5-methylcytosine (5mC), a key epigenetic marker, in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC).
TET2 exhibits activity across various sequence contexts, including CpG, CHH, and CHG sites, whether hemi- or fully methylated. However, the sequences flanking the target 5mC site influence the level of catalytic activity, with a preference for CG contexts. This sequence preference diminishes after the initial oxidation of 5mC to 5hmC.
Although 5mC is predominantly found in CpG sites within mammalian genomes, DNA methylation contexts are more diverse in both invertebrate and plant genomes.
Product Source
An E. coli strain that carries a cloned TET2 gene.
This product is related to the following categories:
- Base Modifying Enzymes
- DNA Methylation Analysis
- DNA Modifying Enzymes & Cloning Technologies
Reagents Supplied
The following reagents are supplied with this product:
| Product | Component | Component # | Stored at | Amount | Concentration |
|---|---|---|---|---|---|
| M1524S | TET2 | M1524SVIAL | -20°C | 1 x 0.05 ml | 8,000 units/ml |
| TET2 Reaction Buffer | B5501SVIAL | -20°C | 1 x 1.25 ml | 5 X | |
| Dithiothreitol (DTT) | B1222AVIAL | -20°C | 1 x 0.5 ml | 100 mM | |
| Fe(II) Solution | B5503SVIAL | -20°C | 1 x 0.25 ml | 500 mM | |
| TET2 Reaction Buffer Supplement | B5502SVIAL | -20°C | 3 x 100 µl | Not Applicable | |
| M1524L | TET2 | M1524LVIAL | -20°C | 1 x 0.25 ml | 8,000 units/ml |
| TET2 Reaction Buffer | B5501SVIAL | -20°C | 2 x 1.25 ml | 5 X | |
| Dithiothreitol (DTT) | B1222AVIAL | -20°C | 2 x 0.5 ml | 100 mM | |
| Fe(II) Solution | B5503SVIAL | -20°C | 1 x 0.25 ml | 500 mM | |
| TET2 Reaction Buffer Supplement | B5502LVIAL | -20°C | 3 x 400 µl | Not Applicable |
Properties & Usage
Materials Required but not Supplied
- Monarch PCR & DNA Cleanup Kit (NEB #T1130)
- Thermolabile Proteinase K (NEB #P8111)
- Nuclease-free Water (NEB #B1500)
Unit Definition
One unit is defined as the amount of enzyme required to modify 1.4 pmol of a 21-mer fluorescently labeled oligonucleotide duplex containing a central methylated CpG moiety in a total reaction volume of 20 µl in 1 hour at 37°C.
Reaction Conditions
1X TET2 Reaction Buffer
Incubate at 37°C
Heat Inactivation
No
Unit Assay Conditions
A fluorescently labeled dsDNA oligonucleotide containing a single 5mC is incubated with 1 unit of TET2 in 1X TET2 Reaction Buffer containing supplement and Fe2+. 1 unit of TET2 must modify ≥80% of 1.75 pmol of substrate as determined by capillary electrophoresis.
Advantages and Features
Features
- Specifically oxidizes 5mC to 5hmC, 5fC, and 5caC in a stepwise manner.
Product Notes
- For enzymes that cannot be heat-inactivated, we recommend using a column for cleanup or treatment with Thermolabile Proteinase K.
- TET2 is active across a wide temperature range (4-80°C). Optimal temperature is 37°C.
- Under nonstandard conditions, TET2 activity can become sensitive to sequence context.
- The methylation status on the opposing strand does not impact TET2 activity.
References
- Crawford D, et al. (2016). Tet2 Catalyzes Stepwise 5-methylcytosine Oxidation by an Iterative and de novo Mechanism. J. Am. Chem. Soc.
- Vaisvila R, et al. (2021). Enzymatic methyl sequencing detects DNA methylation at single-base resolution from picograms of DNA. Genome Research.
- Ravichandran M, et al. (2022). Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function. Science Advances.
- DeNizio J, et al. (2022). TET-TDG active DNA demethylation at CpG and non-CpG site. J Mol Bio.
- WU H, et al. (2014). Reversing DNA Methylation: Mechanisms, Genomics, and Biological Function. Cell.
Protocols, Manuals & Usage
Protocols for this product
- Oxidation of Methylated Cytosine Using TET2 (NEB #M1524)
FAQs & Troubleshooting
FAQs
- Can TET2 (NEB #M1524) be heat inactivated?
- Can the freshly diluted Fe(II) Solution be stored long term?
- Can the reconstituted 1X TET2 Reaction Buffer Supplement be stored longer than 4 months?
- Does TET2 (NEB #M1524) exhibit any sequence preference?
- How long can the lyophilized TET2 Reaction Buffer Supplement be stored?
- Is it normal if the Fe(II) solution is yellow or a color change is observed?
- Is TET2 (NEB #M1524) active on asymmetrically methylated substrates?
- Is TET2 (NEB #M1524) active on methylated RNA?
- Is TET2 (NEB #M1524) active on methylated single stranded DNA?
- Is TET2 (NEB #M1524) inhibited by EDTA?
- Is TET2 (NEB #M1524) inhibited by salt?
- What is the activity of TET2 (NEB #M1524) in other buffers?
- What is the reaction temperature range for TET2 (NEB #M1524)?
Legal and Disclaimers
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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner.
