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.
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 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 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 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.
|DNA Dot Blot||1:1000|
|Methylated DNA IP||1:50|
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.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
Recommended Fluorochrome-conjugated Anti-Mouse secondary antibodies:
NOTE: Cells should be grown, treated, fixed and stained directly in multi-well plates, chamber slides or on coverslips.
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.
posted December 2015
Protocol Id: 865
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.
• 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
• 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.
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
|31482||SimpleDIP™ Cell Lysis Buffer|
|49291||SimpleDIP™ DNA IP Buffer (10X)|
|7009||ChIP Elution Buffer (2X)|
|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|
|51660||5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb|
|98528||Mouse (G3A1) mAb IgG1 Isotype Control (DIP Formulated)|
|86179||SimpleDIP™ Hydroxymethyl Control Spike-In DNA|
|20906||SimpleDIP™ Hydroxymethyl Control Primers|
|7017 / 14654||Magnetic Separation Rack|
|9872||Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile)|
|-||Phenol/Choloroform/Isoamyl Alcohol (25:24:1) Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA|
|-||Chloroform:Isoamyl Alcohol (24:1)|
|-||Taq DNA polymerase|
|-||Real-Time PCR SYBR™ Green Reaction Mix|
|Number of Cells Used||Approximate Yield|
|1 million||6 µg|
|5 million||30 µg|
|10 million||60 µg|
For suspension cells, count cells using a hemocytometer.
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.
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.
NOTE: The 5-Hydroxymethylcytosine (5-hmC) (HMC31) Mouse mAb binds hydroxymethylated 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 hMeDIP 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.
|Reagent||Amount per IP/Input|
|10x SimpleDIP™ DNA IP Buffer||50 µl|
|Sonicated genomic DNA||1 µg|
|SimpleDIP™ Hydroxymethyl Control Spike-In DNA (optional)||1 µl|
|dH2O||Up to 500 µl final volume|
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.
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.
|Primer length||24 nucleotides|
|Amplicon size||150 to 200 bp (for standard PCR) 80 to 160 bp (for real-time quantitative PCR)|
|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|
|a.||Initial Denaturation||95°C||5 min|
|e.||Repeat Steps b-d for a total of 34 cycles.|
|f.||Final Extension||72°C||5 min|
|Reagent||Volume for 1 PCR Reaction (18 µl)|
|Nuclease-free H2O||6 µl|
|5 µM primers||2 µl|
|SYBR™ Green Reaction Mix||10 µl|
|a.||Initial Denaturation||95°C||3 min|
|d.||Repeat step b and c for a total of 40 cycles|
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
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.
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.
|Protocol Step||Issue||Causes and Resolutions|
|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 hydroxymethylated 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 hydroxymethylated 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 hydroxymethylated 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 hydroxymethylated 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 µg 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
Protocol Id: 844
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.
All Species Expected
Monoclonal antibody is produced by immunizing animals with 5-hydroxymethylcytidine.
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).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
SimpleDIP is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.