Anti-5-methylcytosine (5hmC) Antibody

Product

Anti-5-methylcytosine (5hmC) Antibody

Catalog number :AT15113

5-Hydroxymethylcytosine (5hmC) is a DNA pyrimidine nitrogen base derived from cytosine. It is potentially important in epigenetics, because the hydroxymethyl group on the cytosine can possibly switch a gene on and off. It was first seen in bacteriophages in 1952. However, in 2009 it was found to be abundant in human and mouse brains, as well as in embryonic stem cells. In mammals, it can be generated by oxidation of 5-methylcytosine, a reaction mediated by TET enzymes.

Overview

Description: Anti-5-methylcytosine (5hmC) Mouse Monoclonal Antibody

Reactivity: Species independent

Tested applications: MeDIP: 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-hydroxymethylcytosine (5hmC) in single-stranded DNA (so denaturation is required). No cross reactivity with non-methylated cytosine and 5-methylcytosine (5mC) in DNA.

Properties

Immunogen: Recombinant protein fused with 5-Hydroxymethylcytosine.

Applications

WB Image: Western blot analysis of 5-Hydroxymethylcytosine (5hmC) on different conjugations with anti-5-Hydroxymethylcytosine (5hmC) antibody (AT15113) at 1/1,000 dilution.

Lane 1: Uridine-BSA (negative)

Lane 2: Cytidine-BSA (negative)

Lane 3: 5-Methylcytosine-BSA (negative)

Lane 4: 3-Methylcytosine-BSA (negative)

Lane 5: 5-Hydroxymethylcytidine-BSA (positive)

Lysates/proteins at 2 μg/Lane.

Predicted band size: 66 kDa

Observed band size: 66 kDa

ICC/IF Image: Immunocytochemistry analysis of HeLa cells labeling 5-Hydroxymethylcytosine (5hmC) with anti-5-Hydroxymethylcytosine (5hmC) antibody (AT15113) at 1/100 dilution.

Cells were fixed in 70% ethyl alcohol for 5 minutes at room temperature, then subjected to acid hydrolysis using 2M HCl in TBST for 30 minutes at room temperature. Permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and then blocked with 2% BSA for 30 minutes at room temperature. Cells were then incubated with anti-5-Hydroxymethylcytosine (5hmC) antibody (AT15113) at 1/100 dilution in 1% BSA in PBST overnight at 4 ?. Goat Anti-Mouse IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Application Image: MeDIP was performed using anti-5hmC antibody (AT15113) or Normal Mouse IgG. The same amount of unmethylated, 5-Methylcytosine (5mC) or 5-Hydroxymethylcytosine (5hmC) DNA standard 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 recovery, which is equivalent to one.

Anti-5-methylcytosine (5hmC) Antibody

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