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>

Source: Montanera, K. N. et al., Rhee, H. S. <a href="https://app.jove.com/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.

13 Views. Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Immunoprecipitation of Target Protein-DNA Complexes from Motor Neuron Lysate

Transcript