Anti-5-methylcytosine (m5C/5-mC) Antibody

Overview

Description

Anti-5-methylcytosine (m5C/5-mC) Mouse Monoclonal Antibody

Reactivity

Species independent

Tested applications

MeDIP: 5ug-10ug for 1 Reaction
MeRIP: 5ug-10ug for 1 Reaction
Dot blot: 1/1000.
ICC: 1/200 - 1/500.
IHC: 1/100 - 1/500.

Optimal dilutions should be determined by the end user.

Specificity

Reacts to 5-methylcytosine in both single-stranded and double-stranded DNA (But the binding efficiency in double stranded DNA is much lower, so denaturation is required). No cross reactivity with non-methylated cytosine and hydroxymethylcytosine (5-hmC) in DNA. Also reacts to 5-methylcytosine in RNA

Properties

Immunogen

Small Molecule corresponding to 5-methylcytosine (5-mC) conjugated to Bovine Serum Albumin (BSA).

Applications

IHC Image

Immunohistochemical analysis of paraffin-embedded human colon tissue with anti-5-methylcytosine (5-mC) Mouse antibody at 1/200 dilution.

 

The section was denatured in 2M HCl for 15 minutes at room temperature, then neutralized in 0.1M Tris-HCl (pH 8.3) for 10 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

ICC/IF Image

Immunocytochemistry - Anti-5-methylcytosine (5-mC) antibody

 

Immunocytochemical analysis of HeLa cells using two different fixation/denaturation conditions (red); actin filaments have been labeled with fluorescein phalloidin (green), and nuclei stained with DAPI (blue).

 

Chromatin denaturation is required to expose the epitopes in DNA and allow the antibody to efficiently detect 5mC. Stronger denaturing conditions such as HCl (bottom panels) will result in enhanced nuclear staining compared to weaker denaturing conditions such as acetic acid (HAc, top panels).

 

However, stronger denaturants such as using HCl may alter or degrade other molecules and intracellular structures, which can be problematic for experiments involving multi-color staining or looking at subcellular morphology. For those experiments we would suggest using weaker denaturants such as HAc.

Application Figure 1

MeDIP - Anti-5-methylcytosine (5-mC) antibody

 

MeDIP was performed using anti-5-mC antibody. 1 ng of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard (897 bp) was spiked in 1ug of genomic DNA isolated from HeLa cells as the control. Realtime PCR was then performed to determine the capture of DNA standard as in % of input.

Application Figure 2

MeRIP was performed using anti-m5C antibody and Normal Mouse IgG.

The same amount of unmethylated, 5-Methylcytosine (5-mC) RNA standard are immunoprecipitated. Realtime PCR was then performed to determine the capture of RNA standard as in % of INPUT, which is equivalent to one.

Application Figure 3

Dot Blot - Anti-5-methylcytosine (m5C/5-mC) Antibody

 

Dot blot of RNA. The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of  5-Methylcytosine  (5-mC) RNA, and unmethylated RNA. The pre-spotted membrane was then blotted with antibody.