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Index / EpigenTek / MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric / Product Detail : P-1036-96 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric
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#P-1036-96 MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric

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Product name : MethylFlash Hydroxymethylated DNA Quantification Kit Colorimetric

Catalog number : P-1036-96

Quantity: 96 assays

Availability: Yes

Supplier name : EpigenTek

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About this Product :

MethylFlash Hydroxymethylated 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 Hydroxymethylated DNA Quantification Kit Colorimetric .
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More Details about

MethylFlash™ Hydroxymethylated
DNA Quantification Kit
Catalog No. P-1036

Handling
Kit Contents

 

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


Note: The HC3 Negative Control I is an unmethylated polynucleotide containing 20% of 5-cytosine. The HC4 Negative Control II is a methylated polynucleotide containing 20% of 5-methylcytosine. The HC5 Positive Control is a hydroxymethylated polynucleotide containing 20% of hydroxymethylcytosine.

Shipping and Storage


The kit is shipped in two parts: one part at ambient room temperature and the second part on frozen ice packs at 4°C. Upon receipt: (1) Store HC3, HC4, HC5, HC7, and HC8 at –20°C away from light; (2) Store HC1, HC6, HC9, and 8-Well Assay Strips at 4°C away from light; (3) Store remaining components (HC2 and HC10) at room temperature away from light. All components of the kit are stable for 6 months from the date of shipment, when stored properly.

Introduction

Product Use Information


Uses The MethylFlashTM Hydroxymethylated DNA Quantification Kit is suitable for detecting global DNA hydroxymethylation status using DNA isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffinembedded tissues, plasma/serum samples, and body fluid samples. Input DNA The amount of DNA for each assay can be 50-200 ng. For optimal quantification, the input DNA amount should be 200 ng, as hydroxymethylated DNA (hmDNA) is generally less than 0.6% of total DNA.

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: 1-10 ng) or a single quantity of hydroxymethylated DNA can be used as a positive control. Because global hydroxymethylation 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 hydroxymethylated DNA and determine the relative hydroxymethylation 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 Hydroxymethylated DNA Quantification Kit 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.


Product Updates
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 MethylFlashTM Hydroxymethylated DNA Quantification Kit is for research use only and is not
intended for diagnostic or therapeutic application.
Intellectual Property
The MethylFlashTM Hydroxymethylated DNA Quantification Kit and methods of use are covered by
two 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, 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 widely recognized. Quite recently, a novel modified nucleotide called 5-hydroxymethylcytosine (5-hmC) has been detected to be abundant in mouse brains and embryonic stem cells. 5-hydroxymethylcytosine was first seen in bacteriophages in 1952. In mammals, it can be generated by the 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.

Unmethylated DNA Methylated DNA Hydroxymethylated DNA T-C-G-T-C-G-A-C-G T-mC-G-T-mC-G-A-mC-G T-hmC-G-T-hmC-G-A-hmC-G


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 demethylation, chromatin remodeling, and gene expression regulation, specifically in brain-specific gene regulation:
1) Conversion of 5-mC to 5-hmC greatly reduced the affinity of MBD proteins to methylated DNA.
2) The observation that formation of 5-hmC by oxidative damage or by addition of aldehydes via DNMTs prevents DNMT-mediated methylation of the target cytosine.
3) 5-hmC may recruit specific binding proteins that alter chromatin structure or DNA methylation patterns.
4) 5-hmC accounts for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons.

Because of the presence of 5-hmC in DNA with unclear functions in gene regulation and the discovery of the enzymes that produce 5-hmC, it is considered rather important to know the distribution of this base in different cell types and in different compartments of the genome of mammalians. It is particularly important to identify hydroxymethylation status in human cell/tissues with and without diseases. Several chromatography-based techniques such as HPLC and TLC mass spectrometry are used for detecting 5-hmC. However these methods are time consuming and have low throughput with high costs. Currently used methylated DNA analysis methods including restriction enzyme digestion and bisulfite or MeDIP-mediated MS-PCR and sequencing are also not suitable for 5-hmC detection as 5-hmC and 5-mC are virtually indistinguishable with these methods. To address this problem, Epigentek offers the MethylFlash™ Hydroxymethylated DNA Quantification Kit which uses a unique procedure to quantify global DNA hydroxymethylation. The kit has the following advantages and features:
• Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 45 minutes.
• High sensitivity, of which the detection limit can be as low as 40 pg of hydroxymethylated DNA.
• High specificity with no cross-reactivity to unmethylated cytosine and methylcytosine. Only hydroxymethylated DNA (5-hmC) is detected.
• Universal positive and negative controls are included, which are suitable for quantifying hydroxymethylated DNA from any species.
• Strip-well microplate format makes the assay flexible: manual or high throughput analysis.
• Simple, reliable, and consistent assay conditions.

References
1. Robertson KD. Nat Rev Genet. 6:597-610, 2005.
2. Kriaucionis S et al: Science. 324: 929-930, 2009.
3. WYATT GR et al: Biochem J. 55:774-8, 1953.
4. Tahiliani M et al: Science. 324: 930-935, 2009.
5. Valinluck V et al: Nucleic Acids Res. 32: 4100-4108. 2004.
6. Valinluck V et al: Cancer Res. 67:946-50, 2007.
7. Jin SG et al: Nucleic Acids Res. 38: e125, 2010.

Principle and Procedure
The MethylFlashTM Hydroxymethylated DNA Quantification Kit contains all reagents necessary for the quantification of global DNA hydroxymethylation. In this assay, DNA is bound to strip wells that are specifically treated to have a high DNA affinity. The hydroxymethylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The amount of hydroxymethylated DNA is proportional to the OD intensity measured.

 

Usage


Materials Required But Not Supplied
_ Adjustable pipette
_ Aerosol resistant pipette tips
_ Microplate reader capable of reading absorbance at 450 nm
_ 1.5 ml microcentrifuge tubes
_ Incubator for 37°C incubation
_ Plate seal or Parafilm M
_ Distilled water
_ 1X TE buffer pH 7.5 to 8.0
_ Isolated DNA of interest

Protocol
For the best results, please read the protocol in its entirety prior to starting your experiment.

Starting Materials
Input DNA Amount: DNA amount can range from 50 ng to 200 ng per reaction. An optimal amount is 200 ng per reaction. Starting DNA may be in water or in a buffer such as TE.
DNA Isolation: You can use your method of choice for DNA isolation. Epigentek offers a series of genomic DNA isolation kits for your convenience (see page 18 under “Ordering Information”).
DNA Storage: Isolated genomic DNA can be stored at 4°C or –20°C until use.

1.Preparation of 1X Wash Buffer (HC1)
48-Assay Kit: Add 13 ml of HC1 10X Wash Buffer to 117 ml of distilled water (pH 7.2-7.5).
96-Assay Kit: Add 26 ml of HC1 10X Wash Buffer to 234 ml of distilled water (pH 7.2-7.5)
This Diluted HC1 1X Wash Buffer can now be stored at 4°C for up to six months.

2.Preparation of Diluted Positive Control (HC5)
Single Point Control Preparation: Dilute HC5 Positive Control with 1 X TE to 5 ng/6l (1 6l of HC5 + 3 6l of TE).
Standard Curve Preparation: First, dilute HC5 to 10 ng/6l (5 6l of HC5 + 5 6l of 1X TE). Then, prepare six different concentrations with the 10 ng/6l diluted HC5 and 1X TE into 0.0, 0.5, 1.0, 2.0, 5.0, and 10.0 ng/6l according to the following dilution chart:

 

3. DNA Binding
a. Predetermine the number of strip wells required for your experiment. Carefully remove un-needed strip wells from the plate frame and place them back in the bag (seal the bag tightly and store at 4°C).
b. Add 80 6l of HC2 Binding Solution to each well.
c. Add 1 6l of HC3, 1 6l of HC4, 1 6l of Diluted HC5 (see note below), and 200 ng of your Sample DNA (1-8 6l) into the designated wells depicted in Table 1 or Table 2 on Page 15. Mix solution by gently tilting from side to side or shaking the plate several times. Ensure the solution coats the bottom of the well evenly.

Note:
(1) For a single point control, add 1 7l of HC5 at a concentration of 5 ng/7l;
For the standard curve, add 1 7l of diluted HC5 at concentrations of 0.5-10 ng/7l (see the chart in Step 2). The final concentrations should be 0.5, 1, 2, 5, and 10 ng per well.
(2) For optimal binding, sample DNA volume added should not exceed 8 7l.n
d. Cover strip plate with plate seal or Parafilm M and incubate at 37°C for 90 min.
e. Remove the HC2 Binding Solution from each well. Wash each well with 150 6l of the Diluted HC1 1X Wash Buffer each time for three times.

4. Hydroxymethylated DNA Capture
a. Dilute HC6 (at 1:1000 ratio) with the Diluted HC1.
b. Add 50 6l of the Diluted HC6 to each well, then cover and incubate at room temperature for 60 min.
c. Remove the Diluted HC6 solution from each well.
d. Wash each well with 150 μl of the Diluted HC1 each time for three times.
e. Dilute HC7 (at 1:1000 ratio) with the Diluted HC1.
f. Add 50 6l of the Diluted HC7 to each well, then cover and incubate at room temperature for 30 min.
g. Remove the Diluted HC7 solution from each well.
h. Wash each well with 150 μl of the Diluted HC1 each time for four times.
i. Dilute HC8 (at 1:5000 ratio) with the Diluted HC1.
j. Add 50 6l of the Diluted HC8 to each well, then cover and incubate at room temperature for 30 min.
k. Remove the Diluted HC8 solution from each well.
l. Wash each well with 150 μl of the Diluted HC1 each time for five times.

5. Signal Detection
a. Add 100 6l of HC9 to each well and incubate at room temperature for 1 to 10 min away from light. Begin monitoring color change in the sample wells and control wells. The HC9 solution will turn blue in the presence of sufficient hydroxymethylated DNA.
b. Add 50 6l of HC10 to each well to stop enzyme reaction when color in the positive control wells turns medium blue. The color will change to yellow after adding HC10 and the absorbance should be read on a microplate reader at 450 nm within 2 to 15 min.

Note: If the strip-well plate frame does not fit in the microplate reader, transfer the solution to a standard 96-well microplate.

6. 5-hmC Calculation
Relative Quantification: To determine the relative hydroxymethylation status of two different DNA samples, simple calculation can be carried out using the following formula:

S is the amount of input sample DNA in ng.
P is the amount of input positive control (HC5) in ng.
* 5 is a factor to normalize 5-hmC in the positive control to 100%, as the positive control contains only 20% of 5-hmC.


Example calculation:
Average OD450 of HC4 is 0.085
Average OD450 of HC5 is 0.780
Average OD450 of Sample is 0.158
S is 200 ng
P is 5 ng

Absolute Quantification: To quantify the absolute amount of hydroxymethylated DNA using an accurate calculation, first generate a standard curve and plot the OD value versus amount of HC5 at each concentration point. Next, determine the slope (OD/ng) of the standard curve using linear regression (Microsoft Excel’s SLOPE function is suitable for such calculation) using the most linear part (include at least 4 concentration points) of the standard curve for optimal slope calculation. Now calculate the amount and percentage of hydroxymethylated DNA using the following formulas:

S is the amount of input sample DNA in ng.
* 5 is a factor to normalize 5-hmC in the positive control to 100%, as the positive control contains only 20% of 5-hmC.


Example calculation:
Average OD450 of HC4 is 0.085
Average OD450 of sample is 0.158
Slope is 0.14 OD/ng
S is 200 ng

Suggested Strip Well Setup
Table 1. The suggested strip-well plate setup using a single point positive control in a 48-assay format (in a 96-assay format, Strips 7 to 12 can be configured as Sample). The controls and samples can be measured in duplicate.

 

Table 2. The suggested strip-well plate setup for standard curve preparation in a 48-assay format (in a 96-assay format, Strips 7 to 12 can be configured as Sample). The controls and samples can be measured in duplicate.

 

Appendix
Troubleshooting

 
 
 

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