Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit

Antibody-based enrichment of 5-hydroxymethylcytosine (5-hmC) in DNA

 

hMeDIP Kit Overview

hMeDIP Kit
 

The hMeDIP Kit is designed to immunoprecipitate and enrich for DNA fragments containing 5-hydroxymethylcytosine (5-hmC). The hMeDIP Kit contains a highly specific purified 5-hmC antibody and the necessary buffers to perform methylated DNA immunoprecipitation (MeDIP). The affinity of the antibody enables separation of 5-hmC DNA from 5-mC DNA. Additionally, the antibody works to efficiently immunoprecipitate either double-stranded or single-stranded input DNA.

Active Motif's fast, magnetic protocol has been streamlined to minimize the number of wash and incubation steps, saving you valuable time. The kit also includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine DNA controls and PCR primers that can be used to verify the efficiency of the enrichment.


hMeDIP Kit Highlights

  • Specific antibody only enriches 5-hmC methylated DNA
  • Works with either double-stranded or single-stranded DNA
  • Optimized magnetic protocol to minimize hands-on time
  • Includes unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine control DNAs and PCR primers to verify efficiency

FlowChart of hMeDIP

How the ChIC/CUT&RUN Assay Works

Figure 1: Flow chart of the hMeDIP Method.
Fragmented genomic DNA containing 5-hydroxymethylcytosine can be specifically captured from the rest of the genomic DNA with the purified 5-hmC antibody. Following an overnight incubation, the antibody/DNA complex is captured with protein G magnetic beads. Enriched DNA is then eluted from the beads. (Click image to enlarge.)

 

hMeDIP Kit Contents

*Protein G magnetic beads are shipped on dry ice and will arrive frozen. DO NOT refreeze the magnetic beads after their first use. Once thawed, the Protein G magnetic beads should be stored at 4°C.

  • 5-hydroxymethylcytosine pAb (1 μg/μl), 50 μg, Store at -20°C
  • Rabbit IgG (1 μg/μl), 100 μl, Store at -20°C
  • Protease Inhibitor Cocktail (PIC), 100 μl. Store at -20°C
  • Buffer C, 10 ml, Store at -20°C
  • Buffer D, 10 ml, Store at -20°C
  • Elution Buffer AM2, 1.6 ml, Store at -20°C
  • Neutralization Buffer, 1.6 ml, Store at -20°C
  • Hydroxymethylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
  • Methylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
  • Unmethylated APC DNA (50 ng/μl) 10 μl, Store at -20°C
  • APC PCR Primer Mix (2.5 μM) 400 μl, Store at -20°C
  • Protein G magnetic beads* 250 μl, Store at 4°C
  • 0.2 ml PCR tubes 1 pack, Store at RT
  • Bar magnet and glue dots, Store at RT
 

hMeDIP Kit Data

Specificity of 5-hmC enrichment with hMeDIP Kit

Figure 2: Specificity of the hMeDIP Kit.
MseI digested human genomic DNA (500 ng) was spiked with 25 pg of either methylated, hydroxymethylated or unmethylated APC DNA. These samples were then processed using the hMeDIP Kit with the purified 5-hydroxymethylcytosine pAb. Eluted DNA was purified and tested using real time PCR with the included APC PCR primer mix. The 5-hydroxymethylcytosine antibody specifically enriched the IP sample containing the hydroxymethylated APC DNA, but did not enrich for methylated or unmethylated DNA. The APC locus analyzed in this experiment is not methylated in human genomic DNA and therefore should not amplify. This experiment was performed to detect the presence of the spiked control DNA only.  Data shown are the results from samples assayed in duplicate.


 

Fold Enrichment of hydroxymethylcytosine DNA from hMeDIP Kit after normalization with negative control rabbit IgG

Figure 3: Fold enrichment of eluted DNA from 5-hmC IP as compared to negative control IP.
MseI digested human genomic DNA (500 ng) was spiked with 25 pg of either methylated, hydroxymethylated or unmethylated APC DNA. These samples were then processed using the hMeDIP Kit with the purified 5-hydroxymethylcytosine pAb. Eluted DNA was purified and tested using real time PCR with the included APC PCR primer mix. The 5-hydroxymethylcytosine antibody specifically enriched the IP sample containing the hydroxymethylated APC DNA, but did not enrich for methylated or unmethylated DNA. The APC locus analyzed in this experiment is not methylated in human genomic DNA and therefore should not amplify. This experiment was performed to detect the presence of the spiked control DNA only.  The fold enrichment represents the amount of IP DNA recovered from each spike reaction with the 5-hmC antibody normalized against the negative control rabbit IgG IP reaction. Data shown are the results from samples assayed in duplicate.

 

hMeDIP Kit FAQs

How should I prepare libraries for hMeDIP-seq?

The antibody used for hMeDIP works with double-stranded fragments, and so the immunoprecipitation can be done with dsDNA. Any library prep protocol designed for sub-nanomolar levels double-stranded DNA, should work. Our recommendation is to use the NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads.

What are the “spiked” controls used for in the hMeDIP Kit?

The controls consist of a 338 base pair fragment containing 122 cytosine residues from the APC (adenomatosis polyposis coli) gene promoter. The unmethylated, 5-methylcytosine and 5-hydroxymethylcytosine containing versions were created by the inclusion of either unmethylated dCTP, 5-methyl dCTP or 5-hydroxymethyl dCTP. Adding these is an optional step used to verify IP efficiency. Note that in APC locus is not methylated in normal human gDNA. Therefore the only enrichment should be from the “spiked” hMeDNA. These DNA standards are also available separately as catalog no. 55008.

Why is neutralization buffer added after IP?

The Elution Buffer AP4 has a very low pH.

Can the hMeDIP Kit be used to enrich DNA for other marks other than 5hmC?

In a publication by Li, Z., Zhao, S., Nelakanti, R.V. et al., a N6-methyladenine (n6-mA) antibody was used to immunoprecipitate DNA using the Active Motif hMeDIP Kit components, using the alternative denaturing DNA IP protocol. Please see the publication for details.

 

 

hMeDIP Kit Publications

Search our database of customer publications that have used our hMeDIP Kit.


 
 
 

hMeDIP Kit Documents

 

 

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