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MeDIP Kits and Reagents

Immunoenrich for regions of the genome with modified nucleotides

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PhosphoSitePlus® Resource

  • Additional protein information
  • Analytical tools

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REACTIVITY SENSITIVITY MW (kDa) Isotype
All Endogenous Rabbit IgG

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).

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The specificity of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb 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-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb. The top panel shows the antibody only binding to the DNA fragment containing 5-mC, while the bottom panel shows the membrane stained with methylene blue.

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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.

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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 and the SimpleDIP™ Methylated DNA IP (MeDIP) Kit #76853. 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.

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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 datasheet 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

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

SimpleDIP™ Methylated DNA IP (meDIP) Protocol

Required Reagents

Reagents Included

No Name
31482 SimpleDIP™ Cell Lysis Buffer
49291 SimpleDIP™ DNA IP Buffer (10X)
7009 ChIP Elution Buffer (2X)
74252 TE Buffer
89173 3M Sodium Acetate, pH 5.2
9006 ChIP-Grade Protein G Magnetic Beads
10007 DNA Binding Buffer
10008 DNA Wash Buffer (add 4x volume ethanol before use)
10009 DNA Elution Buffer
10010 DNA Purification Columns
10012 Proteinase K
7013 RNase A
28692 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb
75708 Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated)
65822 SimpleDIP™ Human Testis-Specific H2B Promoter Primers
74803 SimpleDIP™ Mouse Intracisternal A-Particle LTR Primers

Reagents Not Supplied

No Name
7017 / 14654 Magnetic Separation Rack
9872 Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile)
12931 Nuclease-free water
- Phenol/Choloroform/Isoamyl Alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA
- Chloroform:Isoamyl Alcohol (24:1)
- Ethanol (96-100%)
- Trypsin
- Taq DNA polymerase
- dNTP mix
- Real-Time PCR SYBR™ Green Reaction Mix

Section I. Genomic DNA Extraction

Before Starting

  • Stimulate or treat 5 million cells for each experiment. This number of cells will generate approximately 30 μg of DNA (30 IPs).
  • Remove and warm SimpleDIP™ Cell Lysis Buffer in a 37°C water bath and ensure SDS is in solution.
Number of Cells Used Approximate Yield
1 million 6 μg
5 million 30 μg
10 million 60 μg
    1. For suspension cells, count cells using a hemocytometer.

    2. For adherent cells, remove media and wash cells with 10 ml ice-cold 1x PBS, completely removing wash from culture dish. Add 2 ml of trypsin to remove the cells from the plate. Add 8 ml of media with serum to neutralize the trypsin after cells are completely detached and mix thoroughly. Count cells using a hemocytometer.

  1. Transfer 5 million cells to a 15 ml conical tube, centrifuge at 250 x g in a bench top centrifuge for 5 min at 4°C. Wash pellet twice with 10 ml ice-cold 1x PBS. Repeat centrifugation after each wash.
  2. Resuspend the cell pellet from step 2 in 500 μl of SimpleDIP™ Cell Lysis Buffer.
  3. Transfer cells and buffer into a 1.5 ml microcentrifuge tube. Add 2 μl of Proteinase K to the tube and incubate overnight (12-18 hr) with shaking at 55°C.
  4. Add 500 μl of phenol/chloroform/isoamyl alcohol (25:24:1) and mix thoroughly by vortexing for 30 sec.
  5. Separate layers by centrifugation at 10,000 x g for 5 min in a microcentrifuge. Carefully transfer the top aqueous layer to a new tube.
  6. Add 500 μl of chloroform/isoamyl alcohol (24:1) to the material and mix thoroughly by vortexing for 30 sec.
  7. Separate layers by centrifugation at 10,000 x g for 5 min in a microcentrifuge. Carefully transfer the top aqueous layer to a new tube.
  8. Add 50 μl of 3M Sodium Acetate, pH 5.2, then 1.0 ml of 100% ethanol chilled at -20°C. Incubate at -20°C overnight or -80°C for 1 hr to precipitate DNA.
  9. Pellet DNA by centrifugation at 10,000 x g for 5 min in a microcentrifuge.
  10. Carefully remove supernatant and wash pellet with 70% ethanol chilled at -20°C. Decant supernatant and air dry or vacuum dry pellet.
  11. Resuspend pellet in 500 μl of TE Buffer and add 2 μl of RNase A. Incubate for 30 min at 37°C.
  12. Repeat steps 5-11 and then resuspend pellet in 200 μl TE Buffer.

Section II. Genomic DNA Shearing and Quantification

  1. Sonicate genomic DNA (from Section I, Step 13) for 5 pulses for 15 sec each at medium setting, keeping tube on ice for 30 sec in between each pulse.

    • Genomic DNA from mouse embryonic stem cells was fragmented to sub 500 bp with 5 sets of 15 sec pulses using a VirTis VIRSONIC 100 Ultrasonic Homogenizer/Sonicator (The VirTis Company, Gardiner, NY) at setting 6 with a 1/8-inch probe.

    • Please see Appendix A for further optimization of sonication conditions.

  2. Remove 5 μl of the genomic DNA from Step 1 and determine DNA fragment size by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be sheared to a length of approximately 100-500 bp.
  3. To determine DNA concentration, transfer 2 μl of genomic DNA from Step 1 to 98 μl TE Buffer to give a 50-fold dilution and read the OD260. The concentration of DNA in μg/ml is OD260 x 2,500. DNA concentration should ideally be between 50 and 150 μg/ml.

Section III. DNA Immunoprecipitation

NOTE: The 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb binds methylated genomic DNA only in the context of single-stranded DNA. However, next-generation sequencing library prep kits require double-stranded DNA for the adaptor ligation step and won’t work efficiently with the heat-denatured DNA from the MeDIP protocol. Therefore, before setting up the DNA immunoprecipitation, the user must perform the adaptor ligation step as recommended by the manufacturer’s DNA library preparation protocol. The user should then use 1 ug of adaptor-ligated DNA for the DNA immunoprecipitation.

Before Starting

  • Remove and warm 10x SimpleDIP™ IP Buffer in a 37°C water bath and ensure there is no precipitate.
  1. In one tube, prepare enough IP mix for the desired number of IPs (see table below). When determining the number of IPs, the user should include the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 sample and 1 additional IP to account for the 10% input. Place mix on ice.
Reagent Amount per IP/Input
10x SimpleDIP™ DNA IP Buffer 50 μl
Sonicated genomic DNA 1 μg
dH2O Up to 500 μl final volume
  1. Remove a 50 μl sample of the diluted DNA and transfer to a microfuge tube. This is the 10% input sample.
  2. For each IP, transfer 500 μl of IP mix to a 1.5 ml microcentrifuge tube and heat each tube for 10 min at 95°C to denature DNA. Be sure to also heat the 10% input. Quickly put samples on an ice water bath for 5 min.

    • From this point forward, it is important to keep all buffers cold and keep samples on ice to maintain single stranded DNA. The input can now be stored at -20°C until further use.

  3. Add 10 μl of 5-Methylcytosine (5-mC) (D3S2Z) Rabbit mAb #28692 or 10 μl of Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 to the appropriate IP samples. Incubate samples overnight at 4°C with rotation.
  4. Resuspend ChIP Grade Protein G Magnetic Beads #9006 by gently vortexing. Immediately Add 20 μl of ChIP Grade Protein G Magnetic Beads #9006 to each sample and incubate for 2 hours at 4°C with rotation. Proceed to Section IV.

Section IV. Washing and Elution of the Immunoprecipitated DNA

Before starting prepare and chill on ice for each IP

  • 4 ml 1x SimpleDIP™ DNA-IP Buffer (400 μl 10x SimpleDIP™ IP Buffer + 3.6 ml water)
  • Remove and warm 2x ChIP Elution Buffer in a 37°C water bath and ensure SDS is in solution.
  • Prepare 150 μl 1X ChIP Elution Buffer (75 μl 2x ChIP Elution Buffer + 75 μl water) for each IP and 10% input sample.
  • Set a water bath or thermomixer to 65°C.
  1. Pellet protein G magnetic beads (from Section III, Step 5) by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove the supernatant.
  2. Add 1 ml of 1x SimpleDIP™ DNA IP buffer to the beads and incubate at 4°C for 5 min with rotation.
  3. Pellet protein G magnetic beads by placing the tubes in a magnetic separation rack. Wait 1 to 2 min for solution to clear and then carefully remove the supernatant.
  4. Repeat Steps 2 and 3 three additional times, for a total of 4 washes.
  5. Add 150 μl of the 1x ChIP Elution Buffer to each IP sample, including the 10% input sample tube. Set aside input sample at room temperature until Step 9.
  6. Elute DNA from the antibody/protein G beads for 30 min at 65°C with gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively elutions can be performed at room temperature with rotation, but may not be as complete.
  7. Pellet protein G magnetic beads by placing the tubes in a magnetic separation Rack and wait 1 to 2 min for solution to clear.
  8. Carefully transfer eluted DNA to a new tube.
  9. Immediately proceed to Section V. Alternatively, samples can be stored -20°C. However, to avoid formation of a precipitate, be sure to warm samples to room temperature before adding DNA Binding Buffer (Section V, Step 1).

Section V. DNA Purification using Spin Columns

Before Starting

  • Add 10 ml of ethanol (96-100%) to DNA Wash Buffer before use. This step only has to be performed once prior to the first set of DNA purifications.
  • Remove one DNA spin column/collection tube for each DNA sample from Section IV.
  1. Add 750 μl DNA Binding Buffer to each DNA sample and vortex briefly.
    • 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
  2. Transfer 450 μl of each sample from Step 1 to a DNA Purification Column in a collection tube.
  3. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  4. Remove the spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  5. Transfer the remaining 450 μl of sample from Step 1 to the spin column in collection tube and repeat steps 3 and 4.
  6. Add 750 μl of DNA Wash Buffer to the spin column in collection tube.
  7. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  8. Remove the DNA spin column from the collection tube and discard the liquid. Replace spin column in the collection tube.
  9. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec.
  10. Discard collection tube and liquid. Retain spin column.
  11. Add 50 μl of DNA Elution Buffer to each DNA spin column and place into a clean 1.5 ml microcentrifuge tube.
  12. Centrifuge at 18,500 x g in a microcentrifuge for 30 sec to elute DNA.
  13. Remove and discard DNA spin column. Eluate is now purified DNA. Samples can be stored at -20°C.

Section VI. Quantification of DNA by PCR

Recommendations

  • Use filter-tip pipette tips to minimize risk of contamination.
  • The control primers included in the kit are specific for genomic regions that should be methylated in most cell types and can be used for standard PCR or quantitative real-time PCR.
  • A hot-start Taq polymerase is recommended to minimize the risk of non-specific PCR products.
  • PCR primer selection is critical. Primers should be designed with close adherence to the following criteria:
Primer length 24 nucleotides
Optimum Tm 60°C
Optimum GC 50%
Amplicon size 150 to 200 bp (for standard PCR) 80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method

  1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be analyzed. These should include the 10% input sample and the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708 and a tube with no DNA to control for DNA contamination.
  2. Add 2 μl of the appropriate DNA sample to each tube.
  3. Prepare a master reaction mix as described below, making sure to add enough reagent for two extra tubes to account for loss of volume. Add 18 μl of master mix to each reaction tube.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free dH2O 12.5 μl
10x PCR Buffer 2.0 μl
4 mM dNTP Mix 1.0 μl
5 μM Primers 2.0 μl
Taq DNA Polymerase 0.5 μl
  1. Start the following PCR reaction program
a. Initial Denaturation 95°C 5 min
b. Denature 95°C 30 sec
c. Anneal 62°C 30 sec
d. Extension 72°C 30 sec
e. Repeat Steps b-d for a total of 34 cycles.
f. Final Extension 72°C 5 min
  1. Remove 10 μl of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the PCR procdut is 102 bp for human TSH2B #65822 and 176 bp for mouse IAP LTR #74803.

Real-Time Quantitative PCR Method

  1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of PCR machine to be used. PCR reactions should include the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (DIP Formulated) #75708, a tube or well with no DNA to control for contamination, and a serial dilution of the 10% input genomic DNA (undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of amplification.
  2. Add 2 μl of the appropriate DNA sample to each tube or well of the PCR plate.
  3. Prepare a master reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of reaction mix to each PCR reaction tube or well.
Reagent Volume for 1 PCR Reaction (18 μl)
Nuclease-free dH2O 6 μl
5 μM primers 2 μl
SYBR™ Green Reaction Mix 10 μl
  1. Start the following PCR Reaction program
a. Initial Denaturation 95°C 3 min
b. Denature 95°C 15 sec
c. Anneal 65°C 60 sec
d. Repeat steps b and c for a total of 40 cycles
  1. Analyze quantitative PCR results using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.

    Percent Input = 10% x 2(C[T] 10%Input Sample – C[T] IP Sample)
    C[T] = CT = Threshold cycle of PCR reaction

Appendix A. Optimization of Sonication Conditions

Optimal conditions for shearing genomic DNA to 150-500 bp in length may depend on cell type and number of cells and the type of sonicator used. Below is a protocol to determine the optimal sonication conditions for a specific cell type and concentration of cells.

  1. Prepare genomic DNA from 5 million cells as described in Section I, Steps 1-13.
  2. Sonicate on medium setting for 10 pulses for 15 sec each, keeping tube on ice for 30 sec in between each pulse. Take a 5 μl aliquot after every 2 pulses. Determine DNA fragment size by electrophoresis in a 1% agarose gel with a 100 bp DNA marker.
  3. Observe which of the sonication conditions produces DNA in the desired range of 150-500 bp. These conditions can then be used in Section II, Step 1 in place of the suggested protocol step.
Genomic DNA from 5 million mouse ES cells

Genomic DNA from 5 million mouse ES cells was fragmented with 0, 2, 4, 6, 8, and 10 sets of 15 sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. DNA samples were then separated by electrophoresis on a 1% agarose gel next to a 100 bp ladder and stained with ethidium bromide.

Appendix B. Troubleshooting Guide

Protocol Step Issue Causes and Resolutions
Genomic DNA Extraction Concentration of fragmented DNA is too low. Not enough cells were added to the genomic DNA extraction. Count a separate plate of cells before performing the genomic DNA extraction to ensure an exact count. The genomic DNA extraction protocol can support up to 10 million cells per 500 ml of SimpleDIP™ Cell Lysis Buffer. Adding more than 10 million cells may inhibit cell lysis and also decrease DNA concentration.
Genomic DNA Shearing and Quantification OD260/280 ratio is lower than 1.8 (impure DNA). Phenol and/or salt carryover occurred during the phenol/chloroform extractions. During the extractions, leave a small amount of the top layer behind ensuring that no phenol or salt is accidentally transferred with the DNA-containing supernatant.
DNA fragments are the incorrect size. Sonication power or the number of pulses was not sufficient to shear the DNA properly. See Appendix A for a DNA shearing optimization protocol.
DNA Immunoprecipitation Can I alter the amount of antibody or DNA used in the IP? The kit was optimized for 1 µg of antibody and 1 µg of genomic DNA. Adding less antibody or DNA may decrease the recovery of methylated DNA, while adding additional antibody or DNA may decrease the specificity of the IP and generate false positive enrichments.
Can other antibodies be used in the kit? The protocol has been validated and optimized for use with the antibody included in the kit. Other antibodies may not perform optimally with the protocol provided in the kit.
Quantification of DNA by PCR Little or no enrichment of methylated DNA In each IP, 1 µg of antibody and 1 µg of genomic DNA should be used. Using less of either may result in decreased recovery of methylated DNA and weaker signal.
The antibody will only bind to single-stranded DNA, so ensure that all protocol steps after denaturation are performed on ice to prevent reannealing.
Incomplete elution of DNA from the beads may decrease recovery of methylated DNA and result in weaker signal. Elution of DNA from protein G beads is optimal at 65°C with frequent mixing to keep beads suspended in solution.
High background in the IgG control immunoprecipitation. In each IP, 1 µg of antibody and 1 µg of genomic DNA should be used. Using additional antibody or DNA may generate higher background by increasing the amount of non-specific interactions. Adding less DNA could cause the signal in the IgG PCR reaction to appear higher relative to your input.
If performing gel-based PCR, scale back on the number of cycles to be sure you are analyzing PCR products within the linear amplification phase of PCR. Otherwise the differences in quantitites of starting DNA cannot be accurately measured. Alternatively, quantify your immunoprecipitations by real-time PCR.
DIP-Sequencing Can this kit be used in sequencing? Yes. However, next-generation sequencing library prep kits require double-stranded DNA for the adaptor ligation step and won’t work efficiently with enriched heat-denatured DNA from the MeDIP protocol. Therefore, before setting up the DNA immunoprecipitation in Section III, the user must perform the adaptor ligation step as recommended by the manufacturer’s DNA library preparation protocol. The user should then use 1 ug of adaptor-ligated DNA for the DNA immunoprecipitation.
Storage When can the protocol be stopped and the material stored until the protocol is ready to be finished? Cell pellets can be flash frozen and stored at -80°C after Section I, Step 2.
Genomic DNA can be stored at -20°C after Section I, Step 9 or Step 13.
Sheared DNA can be stored at -20°C after Section II, Step 3.
DNA IP’s can be stored at -20°C overnight after Section IV, Step 9. However, to avoid formation of precipitate, be sure to warm samples to room temperature before adding DNA Binding Reagent A in Section V, Step1.
IP’d genomic DNA can be stored at -20°C after Section V, Step 13. However, be sure to heat frozen material to 37°C for 10 minutes before use in PCR, as heat treatment releases any DNA bound to the tube during storage.

posted November 2015

Product Usage Information

Application Dilutions
Immunofluorescence (Immunocytochemistry) 1:1600
DNA Dot Blot 1:1000
Methylated DNA IP 1:50

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.

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.

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, 2571-2587.

2.  Turek-Plewa, J. and Jagodziński, P.P. (2005) Cell. Mol. Biol. Lett. 10, 631-647.

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