Global 5-hmC Quantification Kit
Quantify global changes in hydroxymethylcytosine levels
Active Motif's Global 5-hmC Quantification Kit is an ELISA-based assay designed to detect and quantify 5-hydroxymethylcytosine (5-hmC) levels in genomic DNA from different sample types (e.g. normal and diseased), treatment conditions, clinical outcomes or environmental backgrounds. The Global 5-hmC Quantification Kit enables higher throughput processing of samples, requires only nanogram amounts of genomic DNA per well and provides results that are consistent with mass spectrometry quantification.
Assay results for Active Motif's Global 5-hmC Quantification Kit.
Global 5-hmC Quantification Advantages
- Highly sensitive; can detect as little as 0.02% 5-hmC
- Suitable for use with tissue-derived genomic DNA
- Can detect 5-hmC from as little as 10-50 ng of input DNA
- Includes DNA standards for quantification of global 5-hmC levels
- Higher throughput quantification than mass spectrometry
Hydroxymethylation is known to play a role in transcriptional regulation and embryonic development. Research has indicated it may also serve as a prognostic indicator in certain cancers and neurodegenerative disorders. Because gene-specific analysis techniques do not provide information about the global levels of DNA hydroxymethylation within a genome and mass spectrometry methods require large amounts of DNA and provide low throughput analysis, Active Motif developed the Global 5-hmC Quantification Kit. It is a simple, ELISA-based assay to detect and quantify global changes in 5-hydroxymethylcytosine levels. The colorimetric readout is easily quantified using a microplate reader at 450nm.
Active Motif’s Global 5-hmC Quantification Kit contains an optimized protocol and all necessary reagents to perform DNA fragmentation, plate coating, capture, and colorimetric detection of 5-hmC. For added convenience, DNA standards containing known levels of 5-hmC are included in the kit. Standards can be utilized to generate a standard curve for determination of % 5-hmC values for the DNA samples.
To learn more about the Global 5-hmC Quantification Kit, click on the Method, Data, or Contents tabs below. To view a manual or other related documents, click on the Documents tab below.
How does the Global 5-hmC Quantification Kit work?
Figure 1: Schematic of the Global 5-hmC Quantification Kit.
Global 5-hmC Quantification Kit
The Global 5-hmC Quantification Kit is an ELISA-based assay that is designed for detection and quantitation of 5-hydroxymethylcytosine (5-hmC) levels of tissue-derived genomic DNA. DNA standards with known amounts of 5-hmC are provided for generating a standard curve to extrapolate the percentage of 5-hydroxymethylcytosine in the DNA samples.
Figure 1: Comparison of 5-hmC quantification using the Global 5-hmC Quantification Kit and mass spectrometry.
Figure 2: Graphical representation of the standard curve generated with the Global 5-hmC Quantification Kit.
Figure 3: Comparison of various 5-hmC quantification methods.
Contents & Storage
The Global 5-hmC Quantification Kit contains sufficient reagents to perform 96 reactions.
The Global 5-hmC Quantification Kit is shipped on dry ice and contains reagents with multiple storage temperatures inside. Please confirm receipt of all reagents upon arrival and store items at the appropriate temperatures as indicated below. All reagents are guaranteed stable for 6 months from date of receipt when stored properly. This kit includes the following components:
- MseI enzyme (10 U/μl); Store at -20°C
- 10X MseI Reaction Buffer; Store at -20°C
- 5-hmC DNA Standards A, B, C, D, E, F & G (10 ng/μl); Store at -20°C
- DNA Binding Agent (lyophilized); Store at -20°C
- 5-Hydroxymethylcytosine pAb; Store at -20°C
- Anti-rabbit HRP-conj. secondary Ab; Store at 4°C
- Antibody Dilution Buffer; Store at 4°C
- 20X Wash Buffer; Store at 4°C
- Developing Solution; Store at 4°C
- 96-well plate; Store at 4°C
- Stop Solution; Store at RT
- 10X Sample Dilution Buffer; Store at RT
- 10X Buffer W; Store at RT
- Plate sealer; Store at RT
- 0.2 ml PCR stripwell tubes; Store at RT