5-Hydroxymethylcytidine Monoclonal antibody

5-Hydroxymethylcytidine Monoclonal Antibody for Dot Blot, ELISA

Host / Isotype

Mouse / IgG1

Reactivity

chemical compound

Applications

Dot Blot, ELISA

Conjugate

Unconjugated

CloneNo.

1E6C6

Cat no : 68579-1-Ig

Synonyms

hm5C

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Validation Data Gallery

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  ELISA experiment of 5-Hydroxymethylcytidine using 68579-1-Ig

  Indirect ELISA and competitive ELISA results show that this antibody is specific to 5-Hydroxymethylcytidine (hm5C). Indirect ELISA was performed by coating BSA conjugated 5-Hydroxymethylcytidine (hm5C) at 10ng/well followed by blocking with 1% BSA. Serial diluted primary antibody was added to the plates and incubated at 37℃. HRP-goat anti-mouse was used for detection. Competitive ELISA was performed similarly except that different concentration of 5-Hydroxymethylcytidine (hm5C) or its structure analogue compounds are mixed in primary antibody (fixed at 10 ng/mL).

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  Dot Blot experiment of RNA using 68579-1-Ig

  Total RNA was isolated from HeLa cell line and was dotted to NC membrane at different amount as indicated above the dots. The membrane was blocked with BSA and blotted with 5-Hydroxymethylcytidine (hm5C) antibody 68579-1-Ig at 1:5000 followed by incubation of HRP-goat anti-mouse secondary antibody. Signal was developed by ECL substrate. A parallel dot blot was performed using mouse IgG1 isotype control antibody 66360-1-Ig at the same dose.

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Tested Applications

• Positive ELISA detected in: 5-Hydroxymethylcytidine
• Positive Dot Blot detected in: RNA

Recommended dilution

• Enzyme-linked Immunosorbent Assay (ELISA): ELISA : 1:2000-1:20000
• DOT BLOT: DOT BLOT : 1:1000-1:4000

It is recommended that this reagent should be titrated in each testing system to obtain optimal results.

Sample-dependent, Check data in validation data gallery.

Product Information

68579-1-Ig targets 5-Hydroxymethylcytidine in Dot Blot, ELISA applications and shows reactivity with chemical compound samples.

• Tested Reactivity: chemical compound
• Host / Isotype: Mouse / IgG1
• Class: Monoclonal
• Type: Antibody
• Immunogen: PTG
• Full Name: 5-Hydroxymethylcytidine
• Conjugate: Unconjugated
• Form: Liquid
• Purification Method: Protein G purification
• Storage Buffer: PBS with 0.02% sodium azide and 50% glycerol pH 7.3.
• Storage Conditions: Store at -20°C. Stable for one year after shipment. Aliquoting is unnecessary for -20^oC storage. 20ul sizes contain 0.1% BSA.

Background Information

Oxidation of 5-methylcytosine in DNA by ten-eleven translocation (Tet) family of enzymes has been demonstrated to play a significant role in epigenetic regulation in mammals. Recent reaearch shows that Tet enzymes also possess the activity of catalyzing the formation of 5-hydroxymethylcytidine (5-hmrC) in RNA. It is known that RNA carries more than 100 distinct types of modifications, and these modifications modulate the structure and functions of RNA. Ribonucleoside 5-methylcytidine (m5C) is subject to oxidative processing in mammals, forming 5-hydroxymethylcytidine (hm5C) and 5-formylcytidine (f5C). Researchers have identified hm5C in total RNA from all three domains of life and in polyA-enriched RNA fractions from mammalian cells. This suggests m5C oxidation is a conserved process that could have critical regulatory functions inside cells (PMID: 25676849).

Protocol for Dot Blot:
https://www.ptglab.com/protocol/68579-1-IgDotBlot.pdf