5-methylcytosine Monoclonal antibody

5-methylcytosine Monoclonal Antibody for IHC, Dot Blot, ELISA

Host / Isotype

Mouse / IgG2b

Reactivity

5mc, chemical compound, m5c and More (2)

Applications

IHC, IF, Dot Blot, ELISA

Conjugate

Unconjugated

Publications

3

CloneNo.

2C9G9

Reviews (1)

Cat no : 68301-1-Ig

Synonyms

m5C, 5mC

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Validation Data Gallery

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  IHC staining of mouse testis using 68301-1-Ig

  Immunohistochemical analysis of paraffin-embedded mouse testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 10x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

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  IHC staining of mouse testis using 68301-1-Ig

  Immunohistochemical analysis of paraffin-embedded mouse testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

• 

  IHC staining of rat brain using 68301-1-Ig

  Immunohistochemical analysis of paraffin-embedded rat brain tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

• 

  IHC staining of rat testis using 68301-1-Ig

  Immunohistochemical analysis of paraffin-embedded rat testis tissue slide using 68301-1-Ig (5-methylcytosine antibody) at dilution of 1:5000 (under 40x lens). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0).

• 

  ELISA experiment of compound using 68301-1-Ig

  Indirect ELISA and competitive ELISA results show that this antibody is specific to m5C. Indirect ELISA (blue curve, refer to top X-right Y axis) was performed by coating BSA conjugated m5C at 0.4ng/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 m5C or its structure analogue compound Cytidine was mixed in 20ng/mL primary antibody (red curve, refer to bottom X-left Y axis).

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  Dot Blot experiment of HeLa using 68301-1-Ig

  Total DNA 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-methylcytosine antibody 68301-1-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 IgG2b isotype control Monoclonal antibody 66360-3-Ig at the same dose.

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Tested Applications

• Positive IHC detected in: mouse testis tissue, rat brain tissue, rat testis tissue; Note: suggested antigen retrieval with TE buffer pH 9.0; (*) Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0
• Positive ELISA detected in: compound
• Positive Dot Blot detected in: HeLa cells

Recommended dilution

• Immunohistochemistry (IHC): IHC : 1:2500-1:10000
• Enzyme-linked Immunosorbent Assay (ELISA): ELISA : 1:10-1:100
• DOT BLOT: DOT BLOT : 1:2500-1:10000

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.

Published Applications

• IF: See 1 publications below

Product Information

68301-1-Ig targets 5-methylcytosine in IHC, IF, Dot Blot, ELISA applications and shows reactivity with 5mc, chemical compound, m5c samples.

• Tested Reactivity: 5mc, chemical compound, m5c
• Cited Reactivity: human, mouse
• Host / Isotype: Mouse / IgG2b
• Class: Monoclonal
• Type: Antibody
• Immunogen: Compound
• Full Name: 5-methylcytosine
• Conjugate: Unconjugated
• Form: Liquid
• Purification Method: Protein A 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

In eukaryotes, 5-methylcytosine in DNA (5mC) and its oxidized derivatives (5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC)) are the most prominent modifications and have been suggested to contribute to epigenetic gene regulation through a variety of different mechanisms. m5C has also been found in mRNA, rRNA and tRNA of representative organisms from all kinds of species. As reversible epigenetic modifications, m5C modifications of RNA affect the fate of the modified RNA molecules and play important roles in various biological processes including RNA stability control, protein synthesis, and transcriptional regulation (PMID: 35365216). This antibody can recognize both 5mC in DNA and m5C in RNA.

Protocol for Dot Blot:
https://www.ptglab.com/protocol/68301-1-IgDotBlot.pdf

Publications

Species	Application	Title
human		Cancer Lett; NSUN4 mediated RNA 5-methylcytosine promotes the malignant progression of glioma through improving the CDC42 mRNA stabilization; Authors - Zhen Zhao
mouse	IF	Eur J Pharmacol; 5- methylcytidine effectively improves spermatogenesis recovery in busulfan-induced oligoasthenospermia mice; Authors - Chengniu Wang
human		Cancer Biol Ther; Function of m5C RNA methyltransferase NOP2 in high-grade serous ovarian cancer; Authors - Shimin Yang

Reviews

The reviews below have been submitted by verified Proteintech customers who received an incentive for providing their feedback.

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FH

DONGHONG (Verified Customer) (07-02-2024)

I used this antibody for the competitive elisa kit. Coating BSA-m5C and blocking the 96-well plate. Use the the standard to competitive bind m5C antibody. However, the standard curve was not good as m5C from Abcam (ab10805).

• Applications: Other
• Primary Antibody Dilution: 1:1000