5-Methylcytosine antibody

Catalog No. ABIN1385081

Product Details

Target: 5-Methylcytosine

Reactivity: Please inquire

Host: Rabbit

Clonality: Polyclonal

Conjugate: Un-conjugated

Application: Western Blotting (WB), Flow Cytometry (FACS), Immunofluorescence (Paraffin-embedded Sections) (IF (p)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p))

Cross-Reactivity: Human

Cross-Reactivity (Details): 5 MethylCytosine

Purification: Purified by Protein A.

Immunogen: KLH conjugated 5 MethylCytosine

Isotype: IgG

Application Details

Application Notes: IHC-P 1:100-500
IF(IHC-P) 1:50-200

Restrictions: For Research Use only

Independent Validation (IHC)

Validation #104610 (Immunohistochemistry)

by: Prof. Merighi, Laboratory of Neurobiology, Department of Veterinary Sciences, University of Turin

No.: #104610

Date: 07/29/2024

Antigen: 5-Methylcytosine

Lot Number: 9K04V1

Method validated: Immunohistochemistry

Positive Control:

Adult (> 2 months) CD1 mouse brain fixed in 4% paraformaldehyde.

Negative Control:

One control slice for each experimental procedure processed omitting the primary antibody; overnight incubation in diluent solution only.

Notes:

Passed. The antibody works for IHC-P at 1:100 and 1:200 concentrations without microwave antigen retrieval

Validation Images

5-methyl-cytosine immunoreactive neuronal nuclei in the mouse forebrain. A: a group of immunoreactive nuclei in the deep layers of the cerebral cortex (original magnification 20x); B: at higher magnification (40x), the positive 5-methyl-cytosine sites are visible in the form of a punctate reaction. Primary antibody dilution: 1:100. No microwave.

Full Methods

Primary Antibody: ABIN1385081

Full Protocol:

• Perfuse mice with paraformaldehyde 4% in 0.1 M phosphate buffer pH 7.4 and post-fix of samples in the same fixative for an additional 2 h at RT.
• Wash, dehydrate, and embed samples in paraffin wax.
• Wash several times with 0.01 M PBS.
• Cut the brain with a microtome into 6 µm sections and mount them on glass slides.
• After paraffin removal, sections were incubated for 1 hour at room temperature in PBS containing 1% albumin from chicken egg white (Sigma, A5378) and 0.3% Triton-X-100 (BioRad, 161-0407, lot # 00583) to block non-specific binding sites.
• Incubate sections with the primary antibody anti-5-methyl-cytosine (ABIN1385081; Lot 9K04V1) at 1:50, 1:100, and 1:200 in PBS-BSA-(Sigma, A7906)- PLL (Sigma, P1524) at RT in a humid chamber.
• Wash sections 3x 5 min with 0.01 M PBS.
• Incubate sections with anti-rabbit secondary antibody Alexa Fluor 488, 1:500, in 0.1M PBS (Thermo Fisher Scientific, A11034, lot # 2380031) for 1 h at RT.
• Wash sections 3x 5 min with 0.01 M PBS.
• Specimens were mounted in Fluoroshield (Sigma, F6182, lot # MKCB0153V).
• Images were acquired with a Leica DM 6000B fluorescence microscope with a digital camera at 40x magnification.

Handling

Format: Liquid

Concentration: 1 μg/μL

Buffer: 0.01M TBS( pH 7.4) with 1 % BSA, 0.02 % Proclin300 and 50 % Glycerol.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE, which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Storage Comment: Shipped at 4°C. Store at -20°C for one year. Avoid repeated freeze/thaw cycles.

Expiry Date: 12 months

Target Details

Target: 5-Methylcytosine

Alternative Name: 5 MethylCytosine

Target Type: Chemical

Background:

Synonyms: 5-Methyl Cytidine, 5 m C, 5 mC, 5 me C, 5 Me Cytidine, 5 MeCyd, 5 Methyl Cytidine, 5 MethylCytosine, 5-mC, 5-Me Cytidine, 5-Methyl-Cytosine, 5-MethylCytidine.

Background: Cytidine is a nucleoside formed by a cytosine attached to a ribose ring via a beta-N1-glycosidic bond. DNA is methylated on cytidines by DNA methylases (DNMTs)to generate 5-methylcytidine (5-mC), a potent epigenetics marker and regulator of gene expression. The reverse reaction (cytidine demethylation) starts with its oxidation to hydroxymethyl- (5-hmC), formyl- (5-fC), and carboxy- (5-caC) cytidine. Several enzymes, including the Tet family of proteins have been implicated in cytidine demethylation.