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Index / EpigenTek / MethylFlash Methylated DNA Quantification Kit Colorimetric / Product Detail : P-1034-96 MethylFlash Methylated DNA Quantification Kit Colorimetric
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#P-1034-96 MethylFlash Methylated DNA Quantification Kit Colorimetric

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  Price : 749   EUR
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847   CHF

Product name : MethylFlash Methylated DNA Quantification Kit Colorimetric

Catalog number : P-1034-96

Quantity: 96 assays

Availability: Yes

Supplier name : EpigenTek

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About this Product :

MethylFlash Methylated DNA Quantification Kit Colorimetric antibody storage GENTAUR recommends for long therm storage to freeze at -24 C. For short time storage up to 30 days we suggest fridge storage at 1 to 10 C. Prevent multiple freeze taw cycles of MethylFlash Methylated DNA Quantification Kit Colorimetric .
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More Details about

Product Overview

The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC) in a microplate-based format.

This kit is also specifically optimized for paired use with the MethylFlash Hydroxymethylated DNA Quantification Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

Product Features
The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) is a further refinement of our previous Methylamp™ global DNA methylation quantification kits by simplifying the workflow and improving the consistency of results. It uses an innovative method to enable background signals to be extremely low, eliminating the plate drying and blocking steps. Compared to chromatography-based methods such as HPLC or mass spectrometry, this product provides a cost-effective way to accurately measure levels of 5-methylcytosine. 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 4 hours.
  • Innovative kit composition enables background signals to be extremely low, which eliminates the need for plate blocking and allows the assay to be simple, accurate, reliable, and consistent.
  • High sensitivity, of which the detection limit can be as low as 0.2 ng of methylated DNA from just 50 ng of input genomic DNA.
  • Optimized antibody and enhancer solutions allow high specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no or negligible cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA.
  • Universal positive and negative controls are included, which are suitable for quantifying methylated DNA from any species.
  • Strip-well microplate format makes the assay flexible: manual or high throughput analysis.


Fig. 1. Schematic procedure for the MethylFlash™ Methylated DNA Quantification Kit (Colorimetric).

Product Details


Fig. 2. Demonstration of high sensitivity and specificity of methylated DNA detection achieved by the MethylFlash™ kit. Synthetic unmethylated DNA (contains 50% of cytosine) and methylated DNA (contains 50% of 5-methylcytosine) were added into the assay wells at different concentrations and then measured with the MethylFlash™ Methylated DNA Quantification Kit (Colorimetric).


 

Principle & Procedure
The MethylFlash Methylated DNA Quantification Kit (Colorimetric) contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at 450 nm. The amount of methylated DNA is proportional to the OD intensity measured, which can be calculated with the kit's included formulas for relative methylation status of two different DNA samples or absolute quantification of 5-methylcytosine (5-mC) using a standard curve.

Easy, Fast, and Flexible
The entire colorimetric assay has easy-to-follow steps for convenience and speed, which can be completed in 4 hours. The strip-well microplate format allows for a flexible assay in manual or high throughput analysis. Universal positive and negative controls are included with the kit for quantifying methylated DNA from any species such as mammals, plants, fungi, bacteria, and viruses.

Safe and Convenient
All the needed reagents, including negative controls and positive controls, for quantification of global DNA methylation are conveniently packaged in the kit. The direct colorimetric quantification of DNA samples replaces obsolete or inferior methods and eliminates the need for DNA digestion/denaturation, radioactivity, extraction, or chromatography.

Highly Sensitive and Specific
The novel procedure and proprietary kit compositions allow for accurate quantification of methylated DNA to be achieved with high sensitivity and specificity. The detection limit of the input DNA can be as low as 0.2 ng of methylated DNA.

Product Components

ME1 (10X Wash Buffer)
ME2 (Binding Solution)
ME3 (Negative Control, 20 µg/ml)*
ME4 (Positive Control, 20 µg/ml)*
ME5 (Capture Antibody, 1000 µg/ml*
ME6 (Detection Antibody, 400 µg/ml)*
ME7 (Enhancer Solution)*
ME8 (Developer Solution)
ME9 (Stop Solution)
8-Well Assay Strips (With Frame)
User Guide

* Spin the solution down to the bottom prior to use.

Note: The ME3 Negative Control is an unmethylated polynucleotide containing 50% of cytosine. The ME4 Positive Control is a methylated polynucleotide containing 50% of 5-methylcytosine.

Product Citations

Ortiz, R. et. al. Global DNA Methylation in Peripheral Blood mononuclear cells predicts prolonged elevations of plasma Il-6 in Women throughout the Breast Cancer Trajectory. Abstract

Hsu, A. et. al (October 2011). Promoter de-methylation of Cyclin D2 by sulforaphane in prostate cancer cells. Clinical Epigenetics. 3:3. Full Text Article

Ward, A. et. al. (February 2011). Aberrant methylation of Polo-like kinase CpG islands in Plk4 heterozygous mice. BMC Cancer. 11:71. Full PDF Article

Li, W. et. al. (2011). Distribution of 5-hydroxymethylcytosine in different human tissues. J Nucleic Acids. 2011:870726. PubMed Abstract

 

Shipping and Storage
The kit is shipped in two parts: the first part at ambient room temperature and the second part on frozen ice packs at 4°C. Upon receipt: (1) Store ME3, ME4, ME6, and ME7 at –20°C away from light; (2) Store ME1, ME5, ME8, and 8-Well Assay Strips at 4°C away from light; (3) Store remaining components (ME2 and ME9) at room temperature away from light. All components of the kit are stable for 6 months from the date of shipment, when stored properly.
Note: Check ME1 (10X Wash Buffer) contains salt precipitates before use. If so, briefly warm at room temperature or 37°C and shake the buffer until the salts are re-dissolved.


Introduction
Product Use Information
Uses

The MethylFlashTM Methylated DNA Quantification Kit (Colorimetric) is suitable for detecting global DNA methylation status using DNA isolated from any species such as mammalians, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Input DNA The amount of DNA for each assay can be 50 to 200 ng. For optimal quantification, the input DNA amount should be 100 ng, as methylated DNA varies from tissue to tissue and can be less than 1% of total DNA in some species.

Starting Material
Starting materials can include various tissue or cell samples such as cells from flask or microplate cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, body fluid samples, etc.


Internal Control
Both negative and positive DNA controls are provided in this kit. A standard curve can be performed (range: 1 to 10 ng) or a single quantity of methylated DNA can be used as a positive control. Because global methylation can vary from tissue to tissue, and from normal and diseased states, it is advised to run replicate samples to ensure that the signal generated is validated. This kit will allow the user to quantify an absolute amount of methylated DNA and determine the relative methylation states of two different DNA samples.
Precautions
To avoid cross-contamination, carefully pipette the sample or solution into the strip wells. Use aerosolbarrier pipette tips and always change pipette tips between liquid transfers. Wear gloves throughout the entire procedure. In case of contact between gloves and sample, change gloves immediately.


General Product Information
Quality Control
Each lot of the MethylFlashTM Methylated DNA Quantification Kit (Colorimetric) is tested against predetermined specifications to ensure consistent product quality. Epigentek guarantees the performance of all products in the manner described in our product instructions.


Product Warranty
If this product does not meet your expectations, simply contact our technical support unit or your regional distributor. We also encourage you to contact us if you have any suggestions about product performance or new applications and techniques. Safety Suitable lab coat, disposable gloves, and proper eye protection are required when working with this product.


ProductUpdates
Epigentek reserves the right to change or modify any product to enhance its performance and design. The\ information in this User Guide is subject to change at any time without notice. Thus, only use the User Guide that was supplied with the kit when using that kit.


Usage Limitation
The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) is for research use only and is not intended for diagnostic or therapeutic applications.

Intellectual Property
The MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) and methods of use are covered by pending US patents.


A Brief Overview
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5- methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5-mC is also observed in non-CpG contexts. The biological importance of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. For example, global decrease in 5-mC content (DNA hypomethylation) is likely caused by methyl-deficiency due to a variety of environmental influences, and has been proposed as a molecular marker in multiple biological processes such as cancer. It has been well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer. Thus, the quantification of 5-mC content or global methylation in cancer cells could provide very useful information for detection and analysis of this disease. Quite recently, a novel modified nucleotide, 5-hydroxymethylcytosine (5- hmC) has been detected to be abundant in mouse brain and embryonic stem cells. In mammals, it can be generated by oxidation of 5- methylcytosine, a reaction mediated by the Tet family of enzymes and Dnmt proteins. It is a hydroxylated and methylated form of cytosine.

The broader functions of 5-hmC in epigenetics are still a mystery today. However, a line of evidence does show that 5-hmC plays a role in DNA methylation structures and patterns. Because of the presence of both 5-mC and 5-hmC in DNA with possibly different functions, it is important to determine the contents of these two modified nucleotides and their ratios in different cell types and in different compartments of the genome of mammalians. It is particularly important to identify that in healthy and diseased human cell/tissues, the epigenetic change at the DNA level is due to methylation or hydroxymethylation. Several chromatography-based techniques such as HPLC, TLC mass spectrometry are used for detecting 5-mC and 5-hmC. However, these methods are time consuming and have low throughput with high costs. To address this problem, Epigentek offers the MethylFlash™ Methylated DNA Quantification Kit (Colorimetric) to quantify 5-mC or methylated DNA. This kit is optimized for paired use with our MethylFlash™ Hydroxymethylated DNA Quantification Kit for simultaneously quantifying both methylated and hydroxymethylated DNA, or for quantifying methylated DNA by itself. 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 4 hours.
• Innovative kit composition enables background signals to be extremely low, which eliminates the need for plate blocking and allows the assay to be simple, accurate, reliable, and consistent.
• High sensitivity, of which the detection limit can be as low as 0.2 ng of methylated DNA (methylation).
• Optimized antibody and enhancer solutions allow high specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no or negligible cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA.
• Universal positive and negative controls are included, which are suitable for quantifying methylated DNA from any species.
• Strip-well microplate format makes the assay flexible: manual or high throughput analysis.

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