MethylFlash Methylated DNA 5-mC Quantification Kit (Colorimetric)-96 Assays

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    MethylFlash Methylated DNA 5-mC Quantification Kit (Colorimetric)-96 Assays

    We recommend using a newer version of this product: MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric)

    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 an ELISA-like microplate-based format. Compared to LUMA or LINE-1, Alu, and LTR-based assays, MethylFlash™ technology directly quantifies actual global DNA methylation.

    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 and accepts as low as 50 ng of input genomic DNA.
    • Optimized antibody and enhancer solutions allow high specificity to 5mC, 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.

    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 (5mC) 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.

    Comparison of Available ELISA-based Global DNA Methylation Assay Methods 

     MethylFlash(Colorimetric)Competitor ZCompetitor C
    Assay PrincipleDirect ELISADirect ELISAIndirect ELISA
    Format96-well plate96-well plate96-well plate
    SensitivityExcellent detection limit: 0.2 ng of 5-mC DNAGood detection limit: >0.5 ng of 5-mC DNAVery poor detection limit: >1000 ng of 5-mC DNA
    S/N Ratio>20 with very low background <4 with high background N/A*
    Procedural ConvenienceNo need for plate blocking and denaturation of DNARequires plate blocking and DNA denaturationRequires plate blocking, DNA digestion, and antibody competitive incubation in an additional plate
    Protocol Time <4 hr <4 hr >8 hr
    DNA Type & SpeciesUniversal for any species, both ssDNA and dsDNAHuman and mouse ssDNA onlyVarious species
    SpecificitySpecific to 5-mC onlyCross-reaction to 5-hmCN/A*
    Quantitation TypeBoth relative and absolute quantificationRelativeRelative
    Standard ControlStable, quantitative in absolute amount of 5-mC, and can be universally used for any speciesUnstable, cannot be quantitative in amount, and can only be used for human or mouseN/A*
    Minimum Input Amount50 ng100 ng1000 ng
    Accuracy of DetectionHigh, stable and quantified standard control. Proven close correlation with LC-MS analysis by usersUnclear, unstable and un-quantified standard controlN/A*
    % 5-mC calculationSimpleComplicatedNot Provided
    Patented MethodYesNoNo
    PopularityVery high published citation countLow published citation countLow published citation count 
    Support ExpertiseSince 2006Since 2013Since 2011

    * Insufficient information provided by datasheet or product failed to produce data to make conclusive determination