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51660
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb
Primary Antibodies
Monoclonal Antibody

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb #51660

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Citations (2)
5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb: Image 1

DNA immunoprecipitations were performed with 1 μg of genomic DNA from mouse embryonic stem cells and either 10 μl of 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb or 10 μl of Mouse (G3A1) mAb IgG1 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.

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb: Image 2

Confocal immunofluorescent analysis of 293T cells transfected with a construct expressing DDK-tagged TET1 catalytic domain (TET1-CD) using 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb (green) and DYKDDDDK Tag Antibody #2368 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). As expected, 293T cells expressing TET1-CD (red) exhibit inccreased levels of 5-hydroxymethylcytosine (green).

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb: Image 3

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb specificity was determined by dot blot. The same sequence of a 387-base pair DNA fragment was generated by PCR using exclusively unmodified cytosine, 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), 5-carboxylcytosine (5-caC), or 5-formylcytosine (5-fC). The respective DNA fragments were blotted onto a nylon membrane, UV cross-linked, and probed with 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb. The upper panel shows the antibody only binding to the DNA fragment containing 5-hmC, while the lower panel shows the membrane stained with methylene blue.

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb: Image 4

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb specificity 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-hmC.

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb: Image 5

5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb specificity was determined by DNA immunoprecipitation. DNA IPs were performed with genomic DNA prepared from mouse embryonic stem cells, spiked with DNA containing either unmethylated cytosine, 5-methylcytosine (5-mC), or 5-hydroxymethylcytosine (5-hmC). 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 # 51660S
Product # Size Price
51660S
100 µl $ 118

Supporting Data

REACTIVITY All
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgG1

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) 1:400
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)

IMPORTANT: This protocol employs an atypical fixation and denaturation strategy with which only certain targets are compatible. Where appropriate, this protocol will be linked to its validated antibody under the Product Information banner on the product-specific webpage.

A. Solutions and Reagents

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

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1L 1X PBS, add 100 ml 10X Wash Buffer, Phosphate Buffered Saline (#12528) to 900 ml dH2O, mix. Adjust pH to 8.0.
  2. Ethanol , 70% solution, deionized.
  3. 1.5 M Hydrochloric acid.
  4. Blocking Buffer: Purchase ready-to-use Immunofluorescence Blocking Buffer (#12411), or prepare a 1X PBS / 5% normal serum / 0.3% Triton X-100 buffer by adding 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 30 µl Triton X-100 to 9.5 mL 1X PBS. Store at 4°C.
  5. Antibody Dilution Buffer: Purchase ready-to-use Immunofluorescence Antibody Dilution Buffer (#12378), or prepare a 1X PBS / 1% BSA / 0.3% Triton X-100 buffer by adding 0.1 g BSA (#9998) and 30 µl Triton X-100 to 10 mL 1X PBS. Store at 4°C.
  6. Fluorochrome-conjugated Secondary Antibody : Use a secondary antibody that is reactive to your primary antibody host species (e.g., rabbit). Click here for a list of secondary antibodies approved for immunofluorescence.

B. Fixation and Permeabilization

    NOTE: All subsequent incubations should be carried out at room temperature (20-25°C) unless noted otherwise.

  1. Aspirate media then cover specimen with ice-cold 70% ethanol (use enough to cover completely to a depth of 3 - 5 mm, DO NOT LET DRY).
  2. Allow to fix for 5 min.
  3. Rinse three times in 1X PBS for 5 min each.
  4. Aspirate PBS then incubate fixed specimen in 1.5 M HCl for 1 hour.
  5. Rinse two times in 1X PBS for 5 min each.

C. Immunostaining

  1. Block specimen in Blocking Buffer for 60 min.
  2. While blocking, prepare primary antibody in Antibody Dilution Buffer (see product website for recommended dilution range).
  3. Aspirate blocking solution then apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 min each.

  6. NOTE: If using a fluorochrome-conjugated primary antibody, then skip to Section C, Step 8.

  7. Incubate specimen in fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer for 1–2 hours protected from light.
  8. Rinse three times in 1X PBS for 5 min each protected from light.
  9. Counterstain as appropriate.

  10. NOTE: When including fluorescent cellular dyes in your experiment (DNA dyes, etc.), please refer to the dye product page for its recommended protocol. View our listing of cellular dyes validated for use in immunofluorescence.

  11. Mount specimen in an appropriate antifade reagent such as Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold AntiFade Reagent with DAPI (#8961).
  12. For long-term storage, store samples at 4°C protected from light.

posted December 2015

revised December 2020

Protocol Id: 865

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-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb recognizes endogenous levels of 5-hmC; however many cells and tissues contain very low levels of 5-hmC that may fall below the detection limits of this antibody. This antibody has been validated using ELISA, dot blot, and MeDIP assays and shows high specificity for 5-hmC.

Species Reactivity:

All Species Expected

Source / Purification

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

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).

TET protein-mediated cytosine hydroxymethylation was initially demonstrated in mouse brain and embryonic stem cells (5, 8). Since then this modification has been discovered in many tissues, with the highest levels found in the brain (9). While 5-fC and 5-caC appear to be short-lived intermediate species, there is mounting evidence showing that 5-hmC is a distinct epigenetic mark with various unique functions (10,11). The modified base itself is stable in vivo and interacts with various readers including MeCP2 (11,12). The global level of 5-hmC increases during brain development and 5-hmC is enriched at promoter regions and poised enhancers. Furthermore, there is an inverse correlation between levels of 5-hmC and histone H3K9 and H3K27 trimethylation, suggesting a role for 5-hmC in gene activation (12). Lower amounts of 5-hmC have been reported in various cancers including myeloid leukemia and melanoma (13,14).

  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. Kriaucionis, S. and Heintz, N. (2009) Science 324, 929-30.
  9. Globisch, D. et al. (2010) PLoS One 5, e15367.
  10. Gao, Y. et al. (2013) Cell Stem Cell 12, 453-69.
  11. Mellén, M. et al. (2012) Cell 151, 1417-30.
  12. Wen, L. et al. (2014) Genome Biol 15, R49.
  13. Delhommeau, F. et al. (2009) N Engl J Med 360, 2289-301.
  14. Lian, C.G. et al. (2012) Cell 150, 1135-46.

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