Diagenode is excited to introduce its newest products for research of DNA methylation. Diagenode's experience began over 5 years ago with our premier Methylated DNA Immunoprecipitation (MeDIP) kit featuring the monoclonal 33D3 antibody clone. Since that time, Diagenode continues to create the highest quality and broadest range of research products to address methylation studies for the scientific community.

Diagenode latest 5-hydroxymethylation (HMC) products facilitate higher quality detection of the "sixth base" by;


  • Providing the only two monoclonal antibodies available to the research community.
  • Illuminating the precise role of methylation in your research by providing 3 different antibody sources.
  • Providing maximum detection of an extremely rare modification (Only 0.032 % of all nucleotide in mouse embryonic stem cells are hydroxymethylated! ( > read more )
  • Supply You! the researcher with an alternative to bisulfite sequencing and MBD based assays that do NOT distinguish between methylation and hydroxymethylation. ( > read more )
  • Allows You! the scientists to distinguish between methylation and hydroxymethylation by antibody affinity based assay hMeDIP.

 

 


  • hMeDIP kits
    • Immunoprecipitation with 5-hydroxymethylcytosine (5-hmC) or 5-methylcytosine (5-mC) antibodies
    • Include internal and external controls

> hMeDIP kit (mouse monoclonal antibody)
> hMeDIP kit (rat monoclonal antibody)
> hMeDIP kit (rabbit polyclonal antibody)


  • 5-hmC antibodies
    • Highly specific and extensively validated

> 5-hmC monoclonal antibody (mouse) > Sample size available
> 5-hmC monoclonal antibody (rat) > Sample size available
> 5-hmC polyclonal antibody (rabbit) > Sample size available


  • MeDIP kits
    • Immunoprecipitation with 5-hydroxymethylcytosine (5-hmC) or 5-methylcytosine (5-mC) antibodies
    • Include internal and external controls
    • Validated for next generation sequencing

> MagMeDIP kit
> Auto MeDIP kit


  • 5-mC antibodies
    • Highly specific and extensively validated

> 5-mC monoclonal antibody 33D3 (mouse)
> 5-mC monoclonal antibody cl. b (mouse)


hMeDIP results
hMeDIP was performed using the hMeDIP kit (mouse monoclonal antibody). The IgG isotype antibody from mouse was used as negative control. 1 µg of Hela cells DNA was prepared and sonicated with Bioruptor® to obtain DNA fragments of 300-500 bp. Unmethylated, methylated and hydroxymethylated spike-in controls have been used. Finally qPCR using specific primer pairs for the unmethylated, methylated and hydroxymethylated DNA sequences has been performed.


Immunofluorescence results obtained with 5-hmC monoclonal antibody (rat)
Mice NIH-3T3 cells have been grown on coverslips. Cold methanol fixation 10 min. HCl treatment 30 min at 37°C. Neutralisation by borate buffer. Blocking 30 min in PBS-BSA Incubation with 5-hmC monoclonal antibody (rat), dilution 1/1000. Overnight incubation at 4°C. Washes with PBS-BSA. Incubation with secondary anti-rat antibody FITC (1/1500). 45 minutes incubation at room temperature. Washes with PBS. DAPI staining and washing with PBS. Mounting in vectashield.


Dotblot analysis of the Diagenode 5-hmC rabbit polyclonal antibody with the C, mC and hmC PCR controls
100 to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the hmC, mC and C PCR controls from the Diagenode "5-hmC, 5-mC & cytosine DNA Standard Pack" (Cat No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with the rabbit 5-hydroxymethylcytosine polyclonal antibody (dilution 1:200). The membranes were exposed for 30 seconds.


Dotblot analysis of the Diagenode 5-hmC mouse monoclonal antibody with the C, mC and hmC PCR controls
200 to 2 ng (equivalent of 10 to 0.1 pmol of C-bases) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode "5-hmC, 5-mC & cytosine DNA Standard Pack" (Cat No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 2 µg/ml of the mouse 5-hydroxymethylcytosine monoclonal antibody (dilution 1:500). The membranes were exposed for 30 seconds.

 

Bioruptor®


SX-8G IP-Star® Automated System