We wish to thank members of the Brown and Lam labs for their participation in critiquing this video and article. This work was supported by funds from the Canadian Institutes for Health Research and the Michael Smith Foundation for Health Research.

DNA EXTRACTION AND SAMPLE PREPARATION
DNA from a variety of different samples (cultured cells, fresh frozen as well as formalin-fixed paraffin embedded tissues) can be used for MeDIP. It is important to use highly purified DNA without associated proteins such as histones. It is also important to remove as much RNA as possible from the sample, as it can interfere with both DNA quantitation and antibody binding. The quantity of DNA used for MeDIP can range from 200 ng to 1 &micro;g depending on the amount of DNA available. To demonstrate this protocol, 1 &micro;g of DNA will be used. The following protocol will provide high quality double stranded DNA from cultured cells. Other protocols should be employed for extraction of DNA from other sample types.
<ol>
<li>Add 400 &mu;l of digestion buffer to a cell pellet in a 1.7 ml Eppendorf tube.</li>
<li>Add 100 &mu;g of proteinase K to the tube, and incubate overnight at 50&deg;C.</li>
<li>Add 500 &mu;l phenol pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 3 through 5 once.</li>
<li>Add 500 &micro;l 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Add 40 &mu;g of RNase A and incubate 1 hr at 37&deg;C.</li>
<li>Add 500 &mu;l phenol pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 11 through 13 once.</li>
<li>Add 500 &micro;l 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Add 1/10th volume 3 M sodium acetate (40 &mu;l) and mix well.</li>
<li>Add 2 volumes (900 &mu;l) of 100% ethanol, mix well, and place at -20&deg;C for 20 mins.</li>
<li>Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove ethanol, pulse spin, and remove residual ethanol.</li>
<li>Add 500 &mu;l cold 70% ethanol to wash. Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.</li>
<li>Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.</li>
<li>Resuspend DNA in 50 &mu;l of sterilized dH2O overnight at 4&deg;C.</li>
<li>Quantify the DNA using a NanoDrop Spectrophotometer. An A260:A280 ratio of 1.8 is ideal.</li>
<li>Determine DNA quality and size range on an agarose gel with 100 bp ladder. As little as 10 ng of genomic DNA can be run on a 1.7% agarose gel followed by staining using a dye that is highly sensitive to small amounts of DNA such as SYBR Gold.</li>
<li>In one siliconized tube per sample, prepare 1 &micro;g of DNA in a total volume of 50 &mu;l with the remainder of the volume made up with sterilized dH2O.</li>
</ol>
DNA SONICATION
DNA sonication and the MeDIP protocol must be performed in siliconzied tubes to prevent non specific binding of proteins to tube walls. Optimal sonication times for the DNA samples are based on the degree of DNA sample fragmentation as determined from gel electrophoresis (Step 27). For example, DNA extracted from cultured cells should be of very high molecular weight, and will subsequently require more sonication than DNA extracted from archival samples which are often partially degraded. If samples are of uniformly high molecular weight, it is reasonable at this point to proceed with the sonication as described in this protocol without checking each sample individually. If samples are fragmented as with archival samples, it will be necessary to adapt the sonication procedure likely by decreasing sonication times to obtain 300-100bp fragments. If you expect to process samples that are partially degraded, optimization of sonication parameters can be performed on a representative sample from those of interest. Based on the degree of DNA fragmentation observed from gel electrophoresis (Step 27), the experimenter can predetermine the optimal sonication time for the sample. Here we describe a method to obtain 300-1000 bp DNA fragments by sonication with an automated sonicating device (Bioruptor from Diagenode, UCD-200 TM) using high molecular weight DNA.
<ol>
<li>Water in Biorupter must be at 4&deg;C.</li>
<li>Sonicate for 7 mins on automatic settings (30 sec on 30 sec off at maximum power).</li>
<li>Remove 800 ng (40 &mu;l) of sonicated product and place in siliconized 1.7 ml centrifuge tube for the immunoprecipitation (IP) reaction.</li>
<li>Set aside remaining 200 ng (10 &mu;l) to serve as input (IN) reference DNA (store at 4&deg;C).</li>
</ol>
IMMUNOPRECIPITATON OF METHYLATED DNA
<ol>
<li>Denature the DNA that will be used for IP reaction (800 ng) at 95&deg;C for 10 mins in a water bath.</li>
<li>Cool immediately on ice. Let DNA cool completely (approximately 5 mins on ice) before proceeding with next step.</li>
<li>Add 5 &mu;g monoclonal antibody.</li>
<li>Add IP buffer (used at room temperature throughout this protocol) to a final volume of 500 &mu;l.</li>
<li>Incubate for 2 hrs at 4&deg;C in rotating tube holder.</li>
<li>Just before step 5 is complete, prepare Dynabeads by washing (Steps 6-11). First, resuspend the beads thoroughly in the vial by vortexing.</li>
<li>Transfer 30 &mu;l (~ 2 x 107) of resuspended Dynabeads per reaction plus 1, into a new siliconized tube (for example, if doing 8 reactions, remove enough beads for 9, i.e. 270 &mu;l).</li>
<li>Place the tube on the magnetic rack for 2 mins at room temperature.</li>
<li>Pipette off the supernatant. When removing supernatant, avoid touching the beads against inside wall (where the beads attract to the magnet) with the pipette tip.</li>
<li>Remove the tube from the magnet, and resuspend the beads in an excess volume of IP buffer (750 &mu;l-1000 &mu;l). Place the tube back on the magnetic rack for 2 mins at room temperature.</li>
<li>Repeat the wash once more, and then resuspend the washed beads in IP buffer in the original volume removed in Step 7.</li>
<li>Add 30 &mu;l of washed Dynabeads to each IP reaction</li>
<li>Incubate in a rotating tube holder for 2 hrs at 4&deg;C.</li>
<li>After incubation is complete, place the tube on the magnetic rack for 2 mins at room temperature.</li>
<li>Pipette off the supernatant. Avoid touching the inside wall of the tube (where the beads attract to the magnet) with the pipette tip. Add 500 &mu;l of IP buffer. Mix the tube contents and put it back on the magnetic rack for 2 mins. Repeat wash with 500 ul IP buffer one time.</li>
<li>After removing the supernatant from the last wash, resuspend the beads in 400 &mu;l of digestion buffer.</li>
<li>Treat the reaction with 100 &mu;g of Proteinase K and incubate overnight at 50&deg;C.</li>
</ol>
PURIFICATION OF IMMUNOPRECIPITATED DNA
<ol>
<li>Add 500 &mu;l 1:1 phenol/chloroform pH 7 and vortex thoroughly.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 1 through 3 if the interphase between the aqueous and organic layers appears cloudy.</li>
<li>Add 1/10th volume 3 M sodium acetate (40 &mu;l) and vortex.</li>
<li>Add 1 &mu;l of glycogen (20 &mu;g/&mu;l) and vortex.</li>
<li>Add 2 volumes (1000 &mu;l) of 100% ethanol, vortex, and place at -20&deg;C for 20 mins.</li>
<li>Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove ethanol, pulse spin, and remove residual ethanol.</li>
<li>Add 500 &mu;l cold 70% ethanol to wash. Vortex briefly, and spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.</li>
<li>Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.</li>
<li>Resuspend DNA pellet in 10 &mu;l sterilized dH20. </li>
</ol>
VALIDATION BY PCR
<ol>
<li>You may test to ensure that your MeDIP procedure is working by performing the MeDIP protocol using normal human DNA (male or female), and subsequently assaying a region known to be enriched for methylation.</li>
<li>Remove 30% of MeDIP product to PCR tube, and another 30% of MeDIP product to another PCR tube.</li>
<li>Put 10 ng of IN DNA into a PCR tube, and 10 ng of IN DNA into another PCR tube. You should now have four separate PCR reactions to set up.</li>
<li>Perform PCR using H19 and CTRL primers as indicated in Table 1.</li>
<li>Prepare two mastermixes (one for each primer set) for PCR reactions with 12.5 &micro;l total volume as outlined in Table 2.</li>
<li>Thermocycle the PCR using the conditions outlined in Table 3.</li>
<li>Run 5 &micro;l of PCR products on a 2% agarose gel for visualization.</li>
<li>The expected results for successful MeDIP are shown in Table 4.</li>
</ol>
Tables
Table 1: H19 and CTRL primers for PCR validation. 
<table border="1">
<tbody>
<tr>
<td>Primer Set</td>
<td>Forward Primer</td>
<td>Reverse Primer</td>
<td>Anticipated Product Size</td>
</tr>
<tr>
<td>H19</td>
<td>H19_F 5&rsquo;-cgagtgtgcgtgagtgtgag</td>
<td>H19_R 5&rsquo;-ggcgtaatggaatgcttgaa</td>
<td>174 bp</td>
</tr>
<tr>
<td>CTRL (control)</td>
<td>CTRL_F5&rsquo;-gagagcattagggcagacaaa</td>
<td>CTRL_R 5&rsquo;-gttcctcagacagccacattt</td>
<td>139 bp</td>
</tr>
</tbody>
</table>

Table 2. Mastermixes for PCR reactions. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td>H19 Mix (per Rxn)</td>
<td>CTRL Mix (per Rxn)</td>
</tr>
<tr>
<td>ddH2O</td>
<td>6.875</td>
<td>6.875</td>
</tr>
<tr>
<td>10X Buffer</td>
<td>1.25</td>
<td>1.25</td>
</tr>
<tr>
<td>dNTP mix (10 mM each)</td>
<td>0.25</td>
<td>0.25</td>
</tr>
<tr>
<td>MgCl2 (50 mM)</td>
<td>0.25</td>
<td>0.25</td>
</tr>
<tr>
<td>Primers (H19_F/R or CTRL_F/R) (10 &micro;M each F/R)</td>
<td>0.625</td>
<td>0.625</td>
</tr>
<tr>
<td>Platinum Taq</td>
<td>0.25</td>
<td>0.25</td>
</tr>
</tbody>
</table>

Table 3. PCR thermocyling conditions. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td>95&deg;C</td>
<td>5:00</td>
</tr>
<tr>
<td rowspan="3">40X </td>
<td>95&deg;C</td>
<td>0:30</td>
</tr>
<tr>
<td>56&deg;C</td>
<td>0:30</td>
</tr>
<tr>
<td>72&deg;C</td>
<td>0:15</td>
</tr>
</tbody>
</table>

Table 4. Expected PCR results for successful MeDIP. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td colspan="2" align="center">Template DNA</td>
</tr>
<tr>
<td>Primer used</td>
<td>IN</td>
<td>IP</td>
</tr>
<tr>
<td>H19</td>
<td>Positive</td>
<td>Positive</td>
</tr>
<tr>
<td>CTRL</td>
<td>Positive</td>
<td>Negative</td>
</tr>
</tbody>
</table>

<ol>
	<li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+methylome:+approaches+for+global+DNA+methylation+profiling.">The methylome: approaches for global DNA methylation profiling.</a> Trends Genet. 24, 231-237 (2008).</li><li>Lu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics,+disease,+and+therapeutic+interventions.">Epigenetics, disease, and therapeutic interventions.</a> Ageing research reviews. 5, 449-467 (2006).</li><li>Zilberman, D., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+DNA+methylation+patterns.">Genome-wide analysis of DNA methylation patterns.</a> Development. 134, 3959-3965 (2007).</li><li>Feinberg, A. P., Tycko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+history+of+cancer+epigenetics.">The history of cancer epigenetics.</a> Nature reviews. 4, 143-153 (2004).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution,+silencing+potential+and+evolutionary+impact+of+promoter+DNA+methylation+in+the+human+genome.">Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.</a> Nat Genet. 39, 457-466 (2007).</li><li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+and+promoter-specific+analyses+identify+sites+of+differential+DNA+methylation+in+normal+and+transformed+human+cells.">Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.</a> Nat Genet. 37, 853-862 (2005).</li><li>Wilson, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenomics:+mapping+the+methylome.">Epigenomics: mapping the methylome.</a> Cell Cycle. 5, 155-158 (2006).</li><li>Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C. M., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+elaborate+pathway+required+for+Ras-mediated+epigenetic+silencing.">An elaborate pathway required for Ras-mediated epigenetic silencing.</a> Nature. 449, 1073-1077 (2007).</li><li>Karpinski, P., Sasiadek, M. M., Blin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+epigenetic+patterns+in+the+etiology+of+gastrointestinal+cancers.">Aberrant epigenetic patterns in the etiology of gastrointestinal cancers.</a> Journal of applied. 49, 1-10 (2008).</li><li>Maekawa, M., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics:+relations+to+disease+and+laboratory+findings.">Epigenetics: relations to disease and laboratory findings.</a> Current medicinal chemistry. 14, 2642-2653 (2007).</li><li>Vucic, E. A., Brown, C. J., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+of+cancer+progression.">Epigenetics of cancer progression.</a> Pharmacogenomics. 9, 215-234 (2008).</li><li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy.">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429, 457-463 (2004).</li><li>Jones, P. A., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+epigenetic+events+in+cancer.">The fundamental role of epigenetic events in cancer.</a> Nat Rev Genet. 3, 415-428 (2002).</li><li>Fraga, M. F., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation:+a+profile+of+methods+and+applications.">DNA methylation: a profile of methods and applications.</a> Biotechniques. 33, 632-649 (2002).</li></ol>

There is a growing awareness of the significant role DNA methylation plays in disease, therefore the development of assays to measure this modification are becoming increasingly important 3, 12, 13. The MeDIP technique is an amenable tool for screening at both the whole-genome and locus-specific level 6, 7. This technique provides a rapid view of DNA methylation levels using limited amounts of starting DNA and allows for easy comparisons between different sources. Downstream applications using th...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Biorupter sonicator</td>
<td>Tool</td>
<td>Diagenode</td>
<td>UCD-200 TM</td>
<td>&nbsp;</td>
<tr>
<td>1.7ml SafeSeal Microcentrifuge Tubes</td>
<td>Other</td>
<td>Sorenson BioScience</td>
<td>11510</td>
<td>&nbsp;</td>
<tr>
<td>ND 3300 Spectrophotometer</td>
<td>Tool</td>
<td>NanoDrop</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Primary Antibody: Anti-5'-methylcytosine mouse mAb</td>
<td>Reagent</td>
<td>CalBiochem</td>
<td>162 33 D3</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>112-01D</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Tube Rack</td>
<td>Tool</td>
<td>Invitrogen</td>
<td>CS15000</td>
<td>&nbsp;</td>
<tr>
<td>Mini LabRoller</td>
<td>Tool</td>
<td>Labnet International</td>
<td>H5500</td>
<td>&nbsp;</td>
<tr>
<td>IP Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td>
</tr>
<tr>
<td>Digestion Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td>
</tr>		</tbody>
		</table>
		</div>

methylated dna immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

Watch this Scientific Journal Video about Methylated DNA Immunoprecipitation at JoVE.com

Methylated DNA Immunoprecipitation

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

Research

JoVE Journal

Biology

23.2K Views.  BC Cancer Research Centre. This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

Video: Methylated DNA Immunoprecipitation

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

DNA Stable-Isotope Probing (DNA-SIP)

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

DNA Stable-Isotope Probing DNA-SIP

DNA stable-isotope probing is a cultivation-independent method to identify and characterize active communities of microorganisms that are capable of utilizing specific substrates. Assimilation of substrate enriched in heavy isotope leads to incorporation of labelled atoms into microbial biomass. Density gradient ultracentrifugation retrieves labelled DNA for downstream molecular analyses.

DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and ...

IP-FCM:  Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.

The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Immunoprecipitation (ChIP) using Drosophila tissue

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at different developmental stages. Epigenetic regulation contributes to a broad spectrum of biological processes, including cellular differentiation during embryonic development and homeostasis in adulthood. A critical strategy in epigenetic studies is to examine how various histone modifications and chromatin factors regulate gene expression. To address this, Chromatin Immunoprecipitation (ChIP) is used widely to obtain a snapshot of the association of particular factors with DNA in the cells of interest.
ChIP technique commonly uses cultured cells as starting material, which can be obtained in abundance and homogeneity to generate reproducible data. However, there are several caveats: First, the environment to grow cells in Petri dish is different from that in vivo, thus may not reflect the endogenous chromatin state of cells in a living organism. Second, not all types of cells can be cultured ex vivo. There are only a limited number of cell lines, from which people can obtain enough material for ChIP assay.
Here we describe a method to do ChIP experiment using Drosophila tissues. The starting material is dissected tissue from a living animal, thus can accurately reflect the endogenous chromatin state. The adaptability of this method with many different types of tissue will allow researchers to address a lot more biologically relevant questions regarding epigenetic regulation in vivo1, 2. Combining this method with high-throughput sequencing (ChIP-seq) will further allow researchers to obtain an epigenomic landscape.

Chromatin Immunoprecipitation ChIP using Drosophila tissue

Recently high-throughput sequencing technology has greatly increased sensitivity of Chromatin Immunoprecipitation (ChIP) experiment and prompted its application using purified cells or dissected tissue. Here we delineate a method to use ChIP technique with Drosophila tissue, which can address the endogenous chromatin state in a well-characterized biological system.

Epigenetics remains a rapidly developing field that studies how the chromatin state contributes to differential gene expression in distinct cell types at ...

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters within the origin of replication1. Beyond the origin, SeqA is found at the replication forks, where it organizes newly replicated DNA into higher ordered structures2. SeqA associates only weakly with single GATC sequences, but it forms high affinity complexes with DNA duplexes containing multiple GATC sites. The minimal functional and structural unit of SeqA is a dimer, thereby explaining the requirement of at least two GATC sequences to form a high-affinity complex with hemimethylated DNA3. Additionally, the SeqA architecture, with the oligomerization and DNA-binding domains separated by a flexible linker, allows binding to GATC repeats separated by up to three helical turns. Therefore, understanding the function of SeqA at a molecular level requires the structural analysis of SeqA bound to multiple GATC sequences. In protein-DNA crystallization, DNA can have none to an exceptional effect on the packing interactions depending on the relative sizes and architecture of the protein and the DNA. If the protein is larger than the DNA or footprints most of the DNA, the crystal packing is primarily mediated by protein-protein interactions. Conversely, when the protein is the same size or smaller than the DNA or it only covers a fraction of the DNA, DNA-DNA and DNA-protein interactions dominate crystal packing. Therefore, crystallization of protein-DNA complexes requires the systematic screening of DNA length4 and DNA ends (blunt or overhang)5-7. In this report, we describe how to design, optimize, purify and crystallize hemimethylated DNA duplexes containing tandem GATC repeats in complex with a dimeric variant of SeqA (SeqA&Delta;(41-59)-A25R) to obtain crystals suitable for structure determination.

Crystal structure of protein&#x2013;DNA complexes can provide insight into protein function, mechanism, as well as, the nature of the specific interaction. Here, we report how to optimize the length, sequence and ends of duplex DNA for co-crystallization with Escherichia coli SeqA, a negative regulator of replication initiation.

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

Efficient Chromatin Immunoprecipitation using Limiting Amounts of Biomass

Chromatin immunoprecipitation (ChIP) is a widely-used method for determining the interactions of different proteins with DNA in chromatin of living cells. Examples include sequence-specific DNA binding transcription factors, histones and their different modification states, enzymes such as RNA polymerases and ancillary factors, and DNA repair components. Despite its ubiquity, there is a lack of up-to-date, detailed methodologies for both bench preparation of material and for accurate analysis allowing quantitative metrics of interaction. Due to this lack of information, and also because, like any immunoprecipitation, conditions must be re-optimized for new sets of experimental conditions, the ChIP assay is susceptible to inaccurate or poorly quantitative results.
Our protocol is ultimately derived from seminal work on transcription factor:DNA interactions1,2 , but incorporates a number of improvements to sensitivity and reproducibility for difficult-to-obtain cell types. The protocol has been used successfully3,4 , both using qPCR to quantify DNA enrichment, or using a semi-quantitative variant of the below protocol.
This quantitative analysis of PCR-amplified material is performed computationally, and represents a limiting factor in the assay. Important controls and other considerations include the use of an isotype-matched antibody, as well as evaluation of a control region of genomic DNA, such as an intergenic region predicted not to be bound by the protein under study (or anticipated not to show changes under the experimental conditions). In addition, a standard curve of input material for every ChIP sample is used to derive absolute levels of enrichment in the experimental material. Use of standard curves helps to take into account differences between primer sets, regardless of how carefully they are designed, and also efficiency differences throughout the range of template concentrations for a single primer set. Our protocol is different from others that are available5-8 in that we extensively cover the later, analysis phase.

We describe a robust method for chromatin immunoprecipitation using primary T cells. The method is founded on standard approaches, but uses a specific set of conditions and reagents that improve efficiency for limited a quantities of cells. Importantly, a detailed description of the data analysis phase is presented.

Chromatin immunoprecipitation (ChIP) is a  widely-used method for determining the interactions of different proteins with  DNA in chromatin of living ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...