MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric)
For absorbance-based quantitation of global 5fC levels in an ELISA-like format

DNA 5-fC 修飾定量分析試劑

The MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify 5-formylcytosine (5-fC) in a microplate-based format. It is suitable for use with DNA isolated from any species (mammals, plants, fungi, bacteria, viruses, etc.) in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, and body fluid samples.

Background Information

5-formylcytosine (5-fC) has recently been found in mammalian tissue and cells and has been classified as a seventh DNA base. It is formed by the oxidation of 5-hydroxymethylcytosine through TET hydroxylases and has been demonstrated to be an intermediate of the DNA demethylation process. The function of 5-fC in gene regulation is not yet entirely clear, but it has been shown that 5-fC exhibits replication-dependent dilution during mouse pre-implantation development and could be functional in regulating pre-implantation development as a whole. The detection of 5-fC in various tissues and cells is important because 5-fC could be a marker to indicate active DNA demethylation. 5-fC can also be directly excised by Thymine DNA glycosylase (TDG) to allow subsequent base excision repair (BER) processing which converts modified cytosine back to its unmodified state.

Several chromatography-based techniques such as HPLC-MS are used for the detection of 5-fC in tissue and cells. However, these methods are time consuming, require large amounts of DNA and have low throughput with high costs. To address these problems, Epigentek offers the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric), which uses a unique microplate-based procedure to directly quantify 5-fC.

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原理

The MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric) contains all the reagents necessary for the quantification of 5-fC. In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. 5-fC is detected using capture and detection antibodies. The detected signal is enhanced and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The amount of 5-fC is proportional to the OD intensity measured.

適用樣品

The detection limit of the input DNA can be as low as 1 pg of 5-fC with a dynamic range from 10 pg to 400 pg. There is no cross-reactivity to cytosine, 5-mC, or 5-hmC within the indicated concentration range of the sample DNA.

Schematic procedure for the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric).

5fC standard control was added into the assay wells at different concentrations and then measured with the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric).

Demonstration of high specificity of 5fC detection achieved by the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric). Synthetic unmethylated DNA, methylated DNA, hydroxymethylated DNA and 5-formylcytosine DNA standard were added into the assay wells at different concentrations and then measured with the MethylFlash™ 5-Formylcytosine DNA Quantification Kit (Colorimetric).

4- Percentage of 5fC in different tissues measured with the MethylFlash™ 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric).

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產品列表

貨號	產品	數量
P-1041-48	MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric) 5-甲酰胞嘧啶DNA定量試劑套組 (組)(比色法) 廠牌:Epigentek
P-1041-96	MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric) 5-甲酰胞嘧啶DNA定量試劑套組 (組)(比色法) 廠牌:Epigentek

MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric)

• Fast and easy procedure completed within just 3 hours and 45 minutes.
• High sensitivity, of which the detection limit can be as low as 1 pg of 5-fC.
• High specificity with detection of only 5-fC. No cross-reactivity to cytosine, 5-mC, 5-hmC, or 5-caC within the indicated concentration range of the sample DNA.
• Highly convenient assay that uses DNA isolated from cells or tissues directly; no need for DNA digestion or hydrolysis.
• Universal positive and negative controls are included, which are suitable for quantifying 5-fC from any species.
• Strip-well microplate format makes the assay flexible: manual or high throughput analysis.
• Simple, reliable, and consistent assay conditions.

說明書下載

MethylFlash 5-Formylcytosine (5-fC) DNA Quantification Kit (Colorimetric) 說明書