immunoprecipitation of target protein-dna complexes from motor neuron lysate

Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

To begin the chromatin immunoprecipitation, wash the antibody-coated beads in 1 milliliter of blocking solution. Place the tube containing the antibody-coated beads on a rocking platform at 4 degrees Celsius for 5 minutes. After briefly spinning, place the tube on a magnetic rack, and remove the supernatant.
Resuspend the antibody-coated beads in 50 microliters of blocking solution. Add 1 milliliter of sonicated lysates to the antibody-coated beads. Then, incubate the sample on a rocking platform at 4 degrees Celsius overnight. After allowing the sample to incubate overnight, collect liquid from the cap by briefly spinning the tube. Then, place the tube on a magnetic rack, and after 1 minute, remove the supernatant carefully with a pipette.
Next, perform a series of washes as follows. Add 1 milliliter of cold wash buffer containing CPI to the sample. Mix the sample on a rocking platform at 4 degrees Celsius for 5 minutes. Briefly spin the sample tube. Then, place on a magnetic rack. After 1 minute, remove the supernatant with a pipette.

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NOTE: Autoclaved distilled and deionized water (ddH2O) is recommended for making buffers and reaction master mixes.
1. Harvesting and crosslinking cells
<ol>
	<li>After differentiating mouse embryonic stem (ES) cells into postmitotic neurons, add 11% formaldehyde to the harvested cells to a final concentration of 1% (v/v). Rock cells on a rocking platform (a rocker, shaker, or rotator) for 15 min, at room temperature (RT, 25 &#176;C). 
	NOTE: Depending on the target protein to be crosslinked, less formaldehyde crosslinking (for example, a final concentration of 0.5%) or double crosslinking with disuccinimidyl glutarate (DSG) can be used.</li>
	<li>Add 2.5 M glycine to a final concentration of 150 mM to stop the crosslinking reaction. Rock cells on a rocking platform at RT, for 5 min.</li>
	<li>Centrifuge the crosslinked cells in 15 mL conical tubes at RT, for 6 min, at 1,350 x g. Aspirate the solution, then resuspend cells in 5 mL of 1x phosphate-buffered saline (PBS). 
	NOTE: The crosslinked cell pellets can be stored at -80 &#176;C after flash freezing with liquid nitrogen.</li>
</ol>
2. Cell lysis
NOTE: The following steps in this protocol are for approximately 2 x 107 neuronal cells differentiated from mouse ES cells. To break open cells, lysis buffers containing various detergents will be used. Add 50 &#181;L of 1000x complete protease inhibitor (CPI) stock to 50 mL of buffer just prior to use.
<ol>
	<li>Prepare lysis buffers 1&#8722;3 as described in Table 1.</li>
	<li>Thaw crosslinked cell pellets on ice, then thoroughly resuspend cell pellets in 3 mL of lysis buffer 1 in 15 mL conical tubes. Rock at 4 &#176;C for 15 min on a rocking platform. Centrifuge at 1,350 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Thoroughly resuspend pelleted cells in 3 mL of lysis buffer 2. Rock at 4 &#176;C for 10 min on a rocking platform. Centrifuge at 1,350 x g for 5 min at 4 &#176;C and aspirate supernatant.</li>
	<li>Add 1 mL of lysis buffer 3 to each pellet, then keep on ice. Immediately proceed to sonication.</li>
</ol>
3. Sonicating chromatin
NOTE: Keep samples on ice or at 4 &#176;C during this sonication procedure to reduce crosslink reversal.
<ol>
	<li>Using a sterile spatula, add sonication beads (Table of Materials) up to the 0.2 mL graduation mark on 15 mL polystyrene tubes. Wash sonication beads by vortexing in 600 &#181;L of 1x PBS until no dry spots are visible. Centrifuge the tubes for 5 s at 30 x g and aspirate 1x PBS. 
	NOTE: Sonication in polystyrene tubes is more efficient than sonication in polypropylene tubes. If a smaller number of cells (for example, &#60;106 cells) is sonicated, use 1.5 mL polystyrene tubes without adding sonication beads.</li>
	<li>Thoroughly resuspend the nuclear lysates in 1 mL of lysis buffer 3 (from step 2.4), then transfer to the 15 mL polystyrene tubes containing sonication beads. Briefly vortex the polystyrene tubes.</li>
	<li>To fragmentize the crosslinked chromatin DNA, sonicate nuclear lysates at 4 &#176;C for an optimized number of cycles, with power amplitude at 30 W, and sonication cycles set to 30 s on/30 s off. 
	NOTE: Optimization of sonication for each cell type and batch will result in the best chromatin immunoprecipitation-exonuclease (ChIP-exo) yield. For 2 x 107 mouse neuronal cells, 20&#8722;30 sonication cycles at the mentioned settings will fragment the chromatin in the range of 100&#8722;500 bp.</li>
	<li>After sonication is complete, transfer all the supernatant (sonicated lysates), from each sample, to 1.5 mL tubes. Add 10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol) to each sample at a final concentration of 1%. Mix by pipetting.</li>
	<li>To pellet cell debris, centrifuge samples at 13,500 x g for 10 min at 4 &#176;C. Transfer all the sonicated lysates (supernatant) from each sample to new 1.5 mL tubes.</li>
	<li>Take 30 &#181;L of sonicated lysate from each sample (~3% of sonicated lysate) and add to new 1.5 mL tubes to check sonication. Store remaining sonicated lysates at 4 &#176;C until ready for incubation with antibody-coated beads.</li>
</ol>
4. Checking sonication
<ol>
	<li>Make 20 mL of 2x proteinase K buffer by adding 2 mL of 0.5 M Tris-HCl (pH 8.0), 2 mL of 0.5 M ethylenediamine tetraacetic acid (EDTA), 10 mL of 10% sodium dodecyl sulfate (SDS) and 6 mL of autoclaved ddH2O. Do not add CPI to this buffer. Store in 50 mL tube at RT.</li>
	<li>To reverse the protein-DNA crosslink, by removing the proteins, take the 30 &#181;L of sonicated chromatin from each sample (from step 3.6), add 166 &#181;L of autoclaved ddH2O, 200 &#181;L of 2x proteinase K buffer and 4 &#181;L of 20 mg/mL proteinase K. Briefly vortex the samples, and then incubate at 65 &#176;C for 1&#8722;3 h at 24 x g.</li>
	<li>NOTE: The sonicated lysates can be reverse crosslinked at 65 &#176;C overnight.</li>
	<li>Extract DNA using phenol:chloroform:isoamyl alcohol (PCIA: 25:24:1) and ethanol precipitation method as follows. 
	CAUTION: PCIA is toxic. Use under a fume hood with standard personal protective equipment (PPE).
	<ol>
		<li>Add 400 &#181;L of PCIA to each sample in 1.5 mL tubes, set vortex to maximum speed and vortex samples for 20 s. Centrifuge at 18,400 x g for 6 min at RT. Two phases will be observed. Carefully transfer the upper aqueous layer (clear phase) of each sample to new 1.5 mL tubes.</li>
		<li>Add 1 &#181;L of 10 mg/mL RNase A. Incubate samples at 37 &#176;C for 30 min.</li>
		<li>Add 1 &#181;L of 20 mg/mL glycogen to each sample, then precipitate with 1 mL of ice-cold 100% ethanol (stored in a -20 &#176;C freezer). Mix briefly, then incubate samples in -80 &#176;C freezer for 30 min to 1 h. Centrifuge samples at 18,400 x g for 10 min at 4 &#176;C and carefully pour out 100% ethanol.</li>
		<li>Wash pellets with 500 &#181;L of ice-cold 70% ethanol (stored in a -20 &#176;C freezer). Centrifuge at 18,400 x g for 5 min at 4 &#176;C. Carefully pour out 70% ethanol.</li>
		<li>Incubate samples in 1.5 mL tubes at 50 &#176;C until the remaining ethanol is evaporated. Resuspend DNA pellets in 15 &#181;L of autoclaved ddH2O or nuclease-free ddH2O.</li>
		<li>Run the extracted DNA samples, along with a DNA ladder, in a 1.5% agarose gel at 120&#8722;180 V and check the size of the sonicated DNA.</li>
	</ol>
	</li>
</ol>
5. Antibody incubation with beads
NOTE: The following steps in this protocol are for approximately 2 x 107 neuronal cells differentiated from mouse ES cells. Do not freeze and thaw magnetic beads at any point during the ChIP-exo protocol as the beads may crack causing contamination of the sample or the antibody&#39;s performance may be compromised.
<ol>
	<li>After cell lysis and sonication, prepare Protein G magnetic beads (Table of Materials) for ChIP, mix magnetic beads until homogenous, then add 25 &#181;L of magnetic beads to 2 mL protein low bind tubes. 
	NOTE: The type of magnetic beads used will depend on the species of the antibodies.</li>
	<li>Wash beads with 1 mL of blocking solution (Table 1), mix well, then place on a magnetic rack for 1 min. While still on a magnetic rack, remove supernatant once it is clear.</li>
	<li>Add 1 mL of blocking solution to the magnetic beads. Rock tubes for 10 min at 4 &#176;C on a rocking platform. Briefly spin, then place tubes on a magnetic rack and remove supernatant.</li>
	<li>Repeat step 5.3 two more times.</li>
	<li>Add 500 &#181;L of blocking solution to magnetic beads. Briefly spin the antibody against Isl1 (0.04 &#181;g/&#181;L, Table of Materials), then add 4 &#181;g of antibody to corresponding 2 mL protein low bind tubes containing the magnetic beads in blocking solution.</li>
	<li>Repeat step 5.5 without antibody (i.e., the no antibody control for ChIP). 
	NOTE: The amount of antibody to add can be determined empirically by considering the quality of the antibody and the number of cells used for ChIP.</li>
	<li>Rock samples at 4 &#176;C for 6&#8722;24 h on a rocking platform.</li>
</ol>
6. Chromatin immunoprecipitation (ChIP)
<ol>
	<li>Wash antibody-coated beads from step 5.8 with 1 mL of blocking solution. Place samples on a rocking platform at 4 &#176;C for 5 min. Remove supernatant.</li>
	<li>Repeat step 6.1 two more times.</li>
	<li>Resuspend antibody-coated beads in 50 &#181;L of blocking solution, then transfer to new 2 mL protein low bind tubes. For each ChIP sample, add sonicated lysates (~1.0 mL from step 3.6) to antibody-coated beads in 2 mL protein low bind tubes.</li>
	<li>Incubate each sample on a rocking platform overnight at 4 &#176;C.</li>
</ol>
7. ChIP washes
NOTE: Keep samples on ice or at 4 &#176;C to maintain protein-DNA crosslinking during ChIP washes.
<ol>
	<li>Make high salt wash buffer, LiCl wash buffer and 10 mM Tris-HCl buffer (pH 7.4) as described in Table 2. Store in 50 mL tubes at 4 &#176;C. Add 50 &#181;L of 1000x CPI stock to all buffers just prior to use.</li>
	<li>Briefly spin samples in 2 mL protein low bind tubes to collect liquid from the caps, then place on a magnetic rack for 1 min and remove supernatant carefully with a pipette.</li>
	<li>For ChIP washes, add 1 mL of lysis buffer 3 (at 4 &#176;C) to each tube. Mix on a rocking platform at 4 &#176;C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 minute and remove the supernatant with a pipette.</li>
	<li>Add 1 mL of cold high-salt wash buffer to each tube. Mix on a rocking platform at 4 &#176;C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette.</li>
	<li>Add 1 mL of cold LiCl wash buffer to each tube. Mix on a rocking platform at 4 &#176;C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette.</li>
	<li>Add 1 mL of cold 10 mM Tris-HCl buffer (pH 7.4) to each tube. Mix on a rocking platform at 4 &#176;C for 5 min. Briefly spin samples, then place on a magnetic rack for 1 min and remove the supernatant with a pipette. 
	NOTE: Tris-EDTA buffer (pH 8.0) can be used instead of Tris-HCl buffer (pH 7.4).</li>
</ol>
Table 1: Recipes for lysis buffers 1&#8722;3 and blocking buffer. Store in 50 mL tubes at 4 &#176;C. Add 50 &#181;L of 1000x CPI (complete protease inhibitor) stock to all buffers just prior to use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Lysis buffer 1</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>1 M HEPES (pH 7.3)</td>
			<td>2.5</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>1.4</td>
			<td>140 mM</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>0.1</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>50% Glycerol</td>
			<td>10</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>10% Octylphenol ethoxylate</td>
			<td>2.5</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol</td>
			<td>1.25</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Lysis buffer 2</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>0.5 M Tris-HCl (pH 8.0)</td>
			<td>1</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>2</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>0.1</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Lysis buffer 3</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 8.0)</td>
			<td>0.5</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>1</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>0.1</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>10% Deoxycholic Acid</td>
			<td>0.5</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>30% N-Lauroylsarcosine sodium salt solution</td>
			<td>0.83</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Blocking solution</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>250 mg</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Complete Protease Inhibitor (CPI, 1000x)</td>
			<td>50 &#181;L</td>
			<td>1x</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Fill to 50 mL</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Recipes for ChIP washes: High Salt Wash buffer, LiCl Wash buffer and 10 mM Tris-HCl buffer (pH 7.4). Store in 50 mL tubes at 4 &#176;C. Add 50 &#181;L of 1000x CPI stock to all buffers just prior to use.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">High salt wash buffer</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>1 M HEPES (pH 7.3)</td>
			<td>2.5</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>5</td>
			<td>500 mM</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>0.1</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>10% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol</td>
			<td>5</td>
			<td>1%</td>
		</tr>
		<tr>
			<td>5% Deoxycholic acid</td>
			<td>1</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>5% SDS</td>
			<td>1</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">LiCl wash buffer</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>0.5 M Tris-HCl (pH 8.0)</td>
			<td>2</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>0.1</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>1 M LiCl</td>
			<td>12.5</td>
			<td>250 mM</td>
		</tr>
		<tr>
			<td>10% Octylphenol ethoxylate</td>
			<td>2.5</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>5% Deoxycholic acid</td>
			<td>5</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">10 mM Tris-HCl buffer</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Volume (mL)</td>
			<td>[Final]</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl (pH 7.4)</td>
			<td>0.5</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Autoclaved ddH2O</td>
			<td>Fill to 50</td>
			<td></td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose, UltraPure</td>
			<td>Invitrogen</td>
			<td>16500</td>
			<td>Checking Sonication (Section 4.3.6)&#160;</td>
		</tr>
		<tr>
			<td>Albumin, Bovine Serum (BSA), Protease Free, Heat Shock Isolation, Min. 98%</td>
			<td>BioShop</td>
			<td>ALB003</td>
			<td>Blocking Solution</td>
		</tr>
		<tr>
			<td>Antibody against Isl1</td>
			<td>DSHB</td>
			<td>39.3F7</td>
			<td>Antibody incuation with beads (Section 5.6)</td>
		</tr>
		<tr>
			<td>Bioruptor Pico</td>
			<td>Diagenode</td>
			<td>B01060010</td>
			<td>Sonicating Chromatin (Section 3.3)</td>
		</tr>
		<tr>
			<td>Centrifuge 5424 R</td>
			<td>Eppendorf</td>
			<td>5404000138</td>
			<td>Sonicating Chromatin (Section 3.5), Checking Sonication (Section 4.3.1, 4.3.3 and 4.3.4)</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>22623508</td>
			<td>Harvest, cross-linking and freezing cells (Section 1.3), Cell lysis (Section 2.2 and 2.3), Sonicating Chromatin (Section 3.1)</td>
		</tr>
		<tr>
			<td>Complete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>4693159001</td>
			<td>Added to all buffers, except Proteinase K buffer and ChIP Elution buffer</td>
		</tr>
		<tr>
			<td>Deoxycholic Acid Sodium Salt</td>
			<td>fisher scientific</td>
			<td>BP349</td>
			<td>Lysis Buffer 3, High Salt Wash Buffer and LiCl Wash Buffer</td>
		</tr>
		<tr>
			<td>EDTA, 0.5 M, Sterile Solution, pH 8.0</td>
			<td>BioShop</td>
			<td>EDT111</td>
			<td>Lysis Buffer 1-3, Checking Sonication (Section 4.1), High Salt Wash Buffer, LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr>
			<td>Ethyl Alcohol Anhydrous, 100%</td>
			<td>Commercial alcohols</td>
			<td>P006EAAN</td>
			<td>Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr>
			<td>Formaldehyde, 36.5-38%, contains 10-15% methanol</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td>Harvest, cross-linking and freezing cells (Section 1.1)</td>
		</tr>
		<tr>
			<td>Glycerol, Reagent Grade, min 99.5%</td>
			<td>BioShop</td>
			<td>GLY002</td>
			<td>Lysis Buffer 1</td>
		</tr>
		<tr>
			<td>Glycine, Biotechnology Grade, min. 99%</td>
			<td>BioShop</td>
			<td>GLN001</td>
			<td>Harvest, cross-linking and freezing cells (Section 1.2)</td>
		</tr>
		<tr>
			<td>Glycogen, RNA Grade</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
			<td>Checking Sonication (Section 4.3.3)</td>
		</tr>
		<tr>
			<td>HEPES, 1 M Sterile-filtered Solution, pH 7.3</td>
			<td>BioShop</td>
			<td>HEP003</td>
			<td>Lysis Buffer 1, High Salt Wash Buffer</td>
		</tr>
		<tr>
			<td>Lithium Chloride (LiCl), Reagent grade</td>
			<td>Bioshop</td>
			<td>LIT704</td>
			<td>LiCl Wash Buffer</td>
		</tr>
		<tr>
			<td>Magnetic beads for ChIP (Dynabeads Protein G)</td>
			<td>Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td>Dynabeads Protein G (magnetic beads for ChIP)</td>
			<td>Antibody incubation with beads (Section 5)</td>
		</tr>
		<tr>
			<td>Magnetic rack (DynaMag-2 Magnet)</td>
			<td>Invitrogen</td>
			<td>12321D</td>
			<td>Used in many steps in Sections: 5&#160;</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt solution, 30% aqueous solution, &#8805;97.0% (HPLC)</td>
			<td>Sigma</td>
			<td>61747</td>
			<td>Lysis Buffer 3</td>
		</tr>
		<tr>
			<td>Octylphenol Ethoxylate (IGEPAL CA630)</td>
			<td>BioShop</td>
			<td>NON999</td>
			<td>Lysis Buffer 1 and LiCl Wash Buffer</td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol, Biotechnology Grade (25:24:1)</td>
			<td>BioShop</td>
			<td>PHE512</td>
			<td>Checking Sonication (Section 4.3.1)</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS), 1x</td>
			<td>Corning</td>
			<td>21040CV</td>
			<td>Harvest, cross-linking and freezing cells (Section 1.3) and Sonicating Chromatin (Section 3.1), Antibody incuation with beads (Section 5.1)</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td>Checking Sonication (Section 4.3.6)&#160;</td>
		</tr>
		<tr>
			<td>Proteinase K Solution, RNA Grade</td>
			<td>Invitrogen</td>
			<td>25530049</td>
			<td>Checking Sonication (Section 4.2)&#160;</td>
		</tr>
		<tr>
			<td>Rnase A, Dnase and Protease-free</td>
			<td>Thermo Scientific</td>
			<td>EN0531</td>
			<td>Checking Sonication (Section 4.3.2)&#160;</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl), BioReagent</td>
			<td>Sigma</td>
			<td>S5886</td>
			<td>Lysis Buffer 1-3, High Salt Wash Buffer</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS), Electrophoresis Grade</td>
			<td>BioShop</td>
			<td>SDS001</td>
			<td>Checking Sonication (Section 4.1), High Salt Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr>
			<td>Sonication beads and 15 mL Bioruptor Tubes</td>
			<td>Diagenode</td>
			<td>C01020031</td>
			<td>Sonicating Chromatin (Section 3.1 and 3.2)</td>
		</tr>
		<tr>
			<td>ThermoMixer F1.5</td>
			<td>Eppendorf</td>
			<td>5384000020</td>
			<td>Section 4.2, 4.3.2, and 4.3.5</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 7.4</td>
			<td>Sigma</td>
			<td>T2194</td>
			<td>10 mM Tris-HCl Buffer</td>
		</tr>
		<tr>
			<td>Trizma hydrochloride solution (Tris-HCl), BioPerformance Certified, 1 M, pH 8.0</td>
			<td>Sigma</td>
			<td>T2694</td>
			<td>Lysis Buffer 2, Lysis Buffer 3, Checking Sonication (Section 4.1), LiCl Wash Buffer and ChIP Elution Buffer</td>
		</tr>
		<tr>
			<td>VWR Mini Tube Rocker, Variable Speed</td>
			<td>VWR</td>
			<td>10159-752</td>
			<td>Used in many steps in sections: 1, 2, 5, 6 and 7</td>
		</tr>
		<tr>
			<td>2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, Triton X-100, Reagent Grade</td>
			<td>BioShop</td>
			<td>TRX506</td>
			<td>Lysis Buffer 1, Sonicating Chromatin (Section 3.4) and High Salt Wash Buffer</td>
		</tr>
	</tbody>
</table>

Source: Montanera, K. N. et al., Rhee, H. S. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61124">High-Resolution Mapping of Protein-DNA Interactions in Mouse Stem Cell-Derived Neurons using Chromatin Immunoprecipitation-Exonuclease (ChIP-Exo).</a> J. Vis. Exp. (2020)
This video demonstrates the immunoprecipitation of DNA fragments crosslinked with target protein from stem cell-derived motor neuron lysates using target protein-specific antibody-coated magnetic beads.

NOTE: Autoclaved distilled and deionized water (ddH2O) is recommended for making buffers and reaction master mixes.
1. Harvesting and crosslinking cells

	...

Begin with a tube containing pre-treated magnetic beads coated with antibodies specific to a target protein cross-linked to the DNA.&#160;&#160;&#160;
Add a motor neuron lysate containing DNA fragments and incubate with agitation.
The DNA fragments interact with antibody-coated beads, forming bead-DNA complexes.
Centrifuge to collect the liquid from the cap and place it on a magnetic separator to precipitate the bead-DNA complexes.
Remove the supernatant containing unbound DNA fragments.
Introduce a detergent-based lysis buffer to solubilize non-specifically bound DNA fragments.
Centrifuge and perform the magnetic separation, then remove the supernatant.
Wash with a high salt buffer to disrupt non-specific interactions. Centrifuge, perform the magnetic separation, and remove the supernatant.
Wash with LiCl buffer to remove the remaining non-specific interactions. Centrifuge, perform the magnetic separation, and remove the supernatant.
Finally, wash with Tris-HCl buffer, centrifuge, and perform the magnetic separation.
Remove the supernatant and collect the beads containing the target protein-DNA complexes.

15 Views. Immunoprecypitacja docelowych kompleksów białko-DNA z lizatu neuronu ruchowego

Video: Immunoprecypitacja docelowych kompleksów białko-DNA z lizatu neuronu ruchowego - Experiment

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

JoVE Journal

Genetics

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic mechanisms are frequently altered in glioma. Chromatin immunoprecipitation could be used to study the consequences of genetic alterations in glioma that result from changes in histone modifications which regulate chromatin structure and gene transcription. This protocol describes native chromatin immunoprecipitation on murine brain tumor neurospheres.

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory ...

Cancer Research

Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of a wide range of transcription factors (TFs) and cofactors mediating transcriptional activation or repression via changes in chromatin structure. Chromatin immunoprecipitation (ChIP) is a useful molecular biology approach for mapping histone modifications and profiling transcription factors/cofactors binding to DNA, thus providing a snapshot of the dynamic nuclear changes occurring during different biological processes.
To study transcriptional regulation in adipose tissue, samples derived from in vitro cell cultures of immortalized or primary cell lines are often favored in ChIP assays because of the abundance of starting material and reduced biological variability. However, these models represent a limited snapshot of the actual chromatin state in living organisms. Thus, there is a critical need for optimized protocols to perform ChIP on adipose tissue samples derived from animal models.
Here we describe a protocol for efficient ChIP-seq of both histone modifications and non-histone proteins in brown adipose tissue (BAT) isolated from a mouse. The protocol is optimized for investigating genome-wide localization of proteins of interest and epigenetic markers in the BAT, which is a morphologically and physiologically distinct tissue amongst fat depots.

Here we describe a protocol for efficient chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) of brown adipose tissue (BAT) isolated from a mouse. This protocol is suitable for both mapping histone modifications and investigating genome-wide localization of non-histone proteins of interest in vivo.

Most cellular processes are regulated by transcriptional modulation of specific gene programs. Such modulation is achieved through the combined actions of ...

Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Transkrypcja

Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate