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28692
5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb
Primary Antibodies
Monoclonal Antibody

5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692

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Citations (10)
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  1. IF
Immunofluorescence Image 1: 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing DYKDDDDK-tagged TET1 catalytic domain (TET1-CD) using 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb (green) and DYKDDDDK Tag (9A3) Mouse mAb #8146 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). As expected, 293T cells expressing TET1-CD (red) exhibit decreased levels of 5-methylcytosine (green).

Product Image 1: 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb

DNA immunoprecipitations were performed with 1 μg of genomic DNA from NCCIT cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated). The enriched DNA was quantified by real-time PCR using human Aqp2 intron 5 primers, human TIMP3 promoter primers, SimpleDIP™ Human Testis-Specific H2B Promoter Primers, and SimpleChIP® Human GAPDH Exon 1 Primers #5516. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.

Product Image 2: 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb

DNA immunoprecipitations were performed with 1 μg of genomic DNA from mouse embryonic stem cells and either 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated). The enriched DNA was quantified by real-time PCR using mouse Aqp2 exon 1 primers, SimpleDIP™ Mouse Intracisternal-A Particle (IAP) LTR Primers, mouse Lamc3 exon 1 primers, and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.

Product Image 3: 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb was determined by ELISA. The antibody was titrated against a single-stranded DNA oligo containing either unmodified cytosine or differentially modified cytosine (5-mC, 5-hmC, 5-caC, 5-fC). As shown in the graph, the antibody only binds to the oligo containing 5-mC.

Product Image 4: 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb

The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb was determined by DNA immunoprecipitations. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with control DNA containing either unmethylated cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmc). IPs were performed using 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb. The enriched DNA was quantified by real-time PCR using primers specific to the spiked-in control DNA sequence. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input DNA, which is equivalent to one.

To Purchase # 28692S
製品番号 サイズ 価格 在庫
28692S
100 µl

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Rabbit IgG

Application Key:

  • W-Western
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:1600
DNA Dot Blot 1:1000

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Protocol

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

Stock Solutions

B. Specimen Preparation - Cultured Cell Lines (IF-IC)

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Aspirate media, cover cells completely with ice-cold 70% ethanol.
  2. Allow cells to fix for 5 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Add 1.5 M HCl and incubate for 30 minutes at room temperature.
  5. Aspirate HCl and rinse two times in 1X PBS for 5 minutes each.
  6. Proceed with Immunostaining section C.

C. Immunostaining

NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours at room temperature in dark.
  7. Rinse three times in 1X PBS for 5 minutes each.
  8. Mount samples in an appropriate antifade reagent such as Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold AntiFade Reagent with DAPI (#8961).
  9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

posted December 2015

Protocol Id: 864

DNA Dot Blot Protocol

A. Buffers and Reagents

  1. 20X Saline Sodium Citrate (SSC) Buffer: 3.0 M NaCl, 0.3 M Sodium Citrate, pH to 7.0.
  2. 10X SSC Buffer: Dilute 20X SSC buffer 1:2.
  3. 2X DNA Denaturing Buffer: 200 mM NaOH, 20 mM EDTA.
  4. Nuclease-Free Water: (#12931)
  5. Blotting Membrane: This protocol has been optimized for positively charged nylon membranes.
  6. 96-Well Dot Blot Apparatus
  7. 10X Tris Buffered Saline with Tween® 20 (TBST): (#9997) To prepare 1 L 1X TBST: add 100 ml 10X TBST to 900 ml dH2 0, mix.
  8. Nonfat Dry Milk: (#9999)
  9. Blocking Buffer: 1x TBST with 5% w/v nonfat dry milk; for 150 ml, add 7.5 g nonfat dry milk to 150 ml 1X TBST and mix well.
  10. Bovine Serum Albumin (BSA): (#9998)
  11. Primary Antibody Dilution Buffer: 1X TBST with 5% BSA; for 20 ml, add 1.0 g BSA to 20 ml 1X TBST and mix well.
  12. Secondary Antibody Conjugated to HRP: anti-rabbit (#7074); anti-mouse (#7076).
  13. Detection Reagent LumiGLO® chemiluminescent reagent and peroxide (#7003) or SignalFire™ ECL Reagent (#6883)

B. Dot Blot

Note: This protocol is written for spotting fragmented, purified genomic DNA (titration of 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, and 15.625 ng) onto a positively charged nylon membrane using a 96-well dot blotting apparatus. Depending on the source and type of DNA, more or less DNA may be required for detection with the antibody.
Before Starting:
• Purify genomic DNA using a genomic DNA purification protocol or kit and sonicate
genomic DNA to generate fragments between 200 and 500 bp. DNA fragment size
can be analyzed by gel electrophoresis on a 1% agarose gel with a 100 bp DNA
marker.
• Cut a piece of nylon membrane to the size of the dot blot manifold.
• Wet nylon membrane with 10x SSC Buffer.
• Dry membrane by placing it in 96-well dot blot apparatus and applying vacuum.

  1. Dilute fragmented genomic DNA to 100 ng/μl in 100 ul of nuclease-free water.
    Then denature DNA by adding 100 μl of 2X DNA Denaturing Buffer and incubating at 95°C for 10 min.
  2. Add 200 μl of 20X SSC buffer and immediately chill on ice for 5 min.
  3. Add 100 μl of nuclease-free water to bring DNA solution to a final volume of 500 μl with a DNA concentration of 20 ng/μl.
  4. Set up a series of six 2-fold dilutions by adding 250 μl of the DNA solution, starting with the DNA solution in Step 3, to 250 μl of nuclease-free water. This will generate seven DNA samples containing 250 μl DNA at concentrations of 20 ng/μl, 10 ng/μl, 5 ng/μl, 2.5 ng/μl, 1.25 ng/μl, 0.625 ng/μl, and 0.3125 ng/μl.
  5. Apply 50 μl of each of the seven dilution samples into separate wells of the 96-well dot blot apparatus, leaving the last well for nuclease-free water only. The amount of DNA added to each well should then be 1000 ng, 500 ng, 250 ng, 125 ng, 62.5 ng, 31.25 ng, 15.625 ng and 0 ng respectively. Apply gentle vacuum pressure to draw solution through the membrane. Nylon membrane should be mostly dry before step 6.
  6. Remove nylon membrane from the 96-well dot blot apparatus and wrap in plastic wrap.
  7. UV cross-link nylon membrane at 1200 J/m2.

C. Membrane Blocking and Antibody Incubation

  1. Incubate membrane in 25 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  2. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
  3. Incubate membrane and primary antibody (at the appropriate dilution and diluent as recommended in the antibody product datasheet) in 10 ml primary antibody dilution buffer, with gentle agitation overnight at 4°C.
  4. Wash three times for 5 min each with 15 ml of 1X TBST.
  5. Incubate membrane with the species appropriate HRP-conjugated secondary antibody (#7074 Anti-rabbit IgG, HRP-linked Antibody or #7076 Anti-mouse IgG, HRP-linked Antibody) at 1:2000 in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
  6. Wash membrane three times for 5 min each with 15 ml of 1X TBST.
  7. Proceed with detection (Section D)

D. Detection of DNA

  1. Incubate membrane with 10 mL of LumiGLO® (0.5 ml 20x LumiGLO® #7003, 0.5 ml 20x Peroxide, and 9.0 ml purified water) or 10 ml SignalFire™ #6883 (5 ml Reagent A, 5 ml Reagent B) with gentle agitation for 1 min at room temperature.
  2. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10 sec exposure should indicate the proper exposure time.

NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr

posted November 2015

Protocol Id: 804

Specificity / Sensitivity

5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb recognizes endogenous levels of 5-methylcytosine. This antibody has been validated using ELISA, dot blot, and MeDIP assays and shows high specificity for 5-methylcytosine.

Species Reactivity:

All Species Expected

Source / Purification

Monoclonal antibody is produced by immunizing animals with 5-methylcytidine.

Background

Methylation of DNA at cytosine residues is a heritable, epigenetic modification that is critical for proper regulation of gene expression, genomic imprinting, and mammalian development (1,2). 5-methylcytosine is a repressive epigenetic mark established de novo by two enzymes, DNMT3a and DNMT3b, and is maintained by DNMT1 (3, 4). 5-methylcytosine was originally thought to be passively depleted during DNA replication. However, subsequent studies have shown that Ten-Eleven Translocation (TET) proteins TET1, TET2, and TET3 can catalyze the oxidation of methylated cytosine to 5-hydroxymethylcytosine (5-hmC) (5). Additionally, TET proteins can further oxidize 5-hmC to form 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC), both of which are excised by thymine-DNA glycosylase (TDG), effectively linking cytosine oxidation to the base excision repair pathway and supporting active cytosine demethylation (6,7).

Normally DNA methylation occurs in a bimodal fashion, such that CpG dinucleotides are largely methylated across the genome, except in short stretches of CpG-rich sequences associated with gene promoters, known as CpG-islands, where methylation is virtually absent (8). Cancer cell genomes often undergo global hypomethylation, while CpG-islands become hypermethylated, causing their associated promoters to become repressed (9). There is evidence that a number of aberrantly hypermethylated CpG-islands found in carcinomas occur at tumor suppressor genes such as RB1, MLH1, and BRCA1 (10).

  1. Hermann, A. et al. (2004) Cell. Mol. Life Sci. 61, 2571-87.
  2. Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-47.
  3. Okano, M. et al. (1999) Cell 99, 247-57.
  4. Li, E. et al. (1992) Cell 69, 915-26.
  5. Tahiliani, M. et al. (2009) Science 324, 930-5.
  6. He, Y.F. et al. (2011) Science 333, 1303-7.
  7. Ito, S. et al. (2011) Science 333, 1300-3.
  8. Suzuki, M.M. and Bird, A. (2008) Nat Rev Genet 9, 465-76.
  9. Berman, B.P. et al. (2012) Nat Genet 44, 40-6.
  10. Sproul, D. and Meehan, R.R. (2013) Brief Funct Genomics 12, 174-90.

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