Recombinant
RabMAb

Anti-5-carboxylcytosine (5-caC) antibody [RM24 1-A3] (ab185492)

Overview

  • Product name
    Anti-5-carboxylcytosine (5-caC) antibody [RM24 1-A3]
  • Description
    Rabbit monoclonal [RM24 1-A3] to 5-carboxylcytosine (5-caC)
  • Specificity
    We see minimal cross-reactivity with 5fC in the applications tested: <2% in ELISA, 5% in MeDIP, ~20% in nucleotide array. This antibody will potentially detect 5caC in RNA. If that is present in your sample, we recommend using +/- RNase treatment as a control.
  • Tested applications
    Suitable for: Nucleotide Array, ELISA, MeDIPmore details
  • Immunogen

    Chemical/ Small Molecule. 5-carboxylcytidine conjugated to keyhole limpet haemocyan (KLH).

  • General notes

    This product is a recombinant rabbit monoclonal antibody.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents

Properties

Applications

Our Abpromise guarantee covers the use of ab185492 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Nucleotide Array Use a concentration of 1 µg/ml.
ELISA Use a concentration of 0 - 1 µg/ml.
MeDIP Use a concentration of 5 - 0.1 µg/ml.

100 µL of sheep anti-rabbit magnetic beads were saturated with 5 µg of ab185492. Then 15 µL of this antibody-bead complex was combined with 12 µg of the corresponding input DNA.

Target

  • Alternative names
    • 5 c C antibody
    • 5 ca C antibody

Images

  • ab185492  was tested using an Indirect ELISA approach. The wells were coated with nucleotide conjugated to BSA (1µg/ml at 100µl/well) overnight at 4°C, followed by a 5% BSA blocking step for 1 hour at room temperature. The primary antibody was then added at a dilution range of  0 -1000 ng/ml (100µl/well) for 1hr at room temperature. A HRP-conjugated anti-rabbit IgG (heavy and light chain) was used as a secondary antibody at 1:20,000 dilution for 1hr at room temperature. ab185492 has 1.5% cross reactivity with 5fC and less than 1% with all other control peptides, when compared with it's reactivity with the immunogen 5caC.

  • Input DNAs was prepared by TdT mediated extension of 19 nt DNA oligonucleotide carrying 5’ biotin tag. The extension was carried out in the presence of equimolar mixture of dATP, dTTP, dGTP and corresponding modified dCTP. Following overnight enzymatic reaction the DNA was purified by ethanol precipitation and reconstituted in TE buffer. 5’ biotin tag on the resulting DNA molecules enables their quantification by streptavidin-HRP in the downstream immunoprecipitation process.

    100 µL of Sheep-anti-Rabbit magnetic beads were saturated with 5µg of ab185492 (blue) or negative control (rabbit IgG isotype control, ab172730, in red) in 1% BSA/TBS/0.1% Triton X-100. No antibody was added to the beads-only control (yellow).

    Equivalent of 15 µL antibody-beads complex or control beads was combined with 12 µg of the corresponding input DNA in TBS/0.1% Triton X-100 (100 µL final volume). Following 1 hour incubation at room temperature the beads were washed three times with TBS/0.1% Triton X-100. Next, the beads were resuspended in 100 µL of streptavidin-HRP (ab7403) diluted to 1 µg/mL in 50 mM Tris pH8, 650 mM NaCl, 0.1% Triton X-100. After 1 hour of incubation at room temperature the beads were washed four times with TBS/0.1% Triton X-100. To quantify the immunoprecipitated DNA the beads were resuspended in 100 µL of Optiblot ECL mixture (ab133406) followed by chemiluminescence capture on CCD camera and images analysis in ImageJ.

  • BSA conjugates of non-modified cytosine along with 5-mC, 5-hmC, 5-fC, 5-caC were deposited onto nitrocellulose-covered glass slides and allowed to dry. Six dilutions of each conjugate were printed in quadruplicate. The slides were blocked with 2% BSA/TBST and then incubated for 2 h in ab185492 diluted to 1.0 µg/mL in the blocking buffer. Following washes, Alexa-647 labelled goat anti rabbit secondary antibody was used to detect binding of ab185492.

     

References

This product has been referenced in:
  • Marina RJ  et al. TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing. EMBO J 35:335-55 (2016). Read more (PubMed: 26711177) »

See 1 Publication for this product

Customer reviews and Q&As

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Embryonic stem cells)
Permeabilization
Yes - 0.5% TX100
Specification
Embryonic stem cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: rt°C
Fixative
Paraformaldehyde
Username

Dr. Fatima Santos

Verified customer

Submitted Feb 05 2016

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up