Name: chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae
Uploaded: 2017-12-29T15:00:00
Description: Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Los autores desean agradecer a miembros del laboratorio verde para discusiones útiles. Este trabajo fue apoyado en parte por subvenciones de NIH R03AG052018 y R01GM124342 a E.M.G.

1. atar los anticuerpos bolas magnéticas
<ol>
	<li>Mezcla bien las bolas magnéticas de proteína A/G y transferir 20 μl de granos magnéticos por una inmunoprecipitación (IP) muestra a un tubo de 1,5 mL. Nota: Cuando el pipeteo granos, utilizar puntas de ancha, baja retención.</li>
	<li>Coloque el tubo con los granos en un soporte magnético y permitir cuentas recoger en el lado del tubo. Eliminar el sobrenadante.</li>
	<li>Lavar los granos 3 veces con 1 mL de solución salina fría tamponada con Tris (TBS) (50...

<ol>
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El procedimiento aquí descrito permite la eficiente recuperación de ADN asociada con histonas modificadas en las células de levadura por la inmunoprecipitación. Esto es seguido por qPCR utilizando cebadores que amplifican regiones de interés para determinar el enriquecimiento local o el agotamiento de las modificaciones específicas de histonas. A pesar de ser desarrollado como un método hace casi 20 años, chIP sigue siendo el ensayo definitorio para investigar el estado de modificación de las histonas en diferen...

La modificación poste-de translación dinámica (PTM) de las histonas es un mecanismo regulador fundamental para muchos procesos con plantilla de ADN, incluyendo transcripción, replicación y reparación de ADN1,2. La capacidad para determinar la abundancia y la localización precisa de las histonas modificadas concomitantes con estos procesos por lo tanto es fundamental para entender la regulación bajo diferentes condiciones en la célula. El desarrollo de inmunoprecipitación de cromatina (chIP) como un método en gran parte provino de los estudios bioquímicos de las interacciones de proteínas con el ADN, particularmente en vitro métodos reticulantes químicos, junto con la necesidad de evaluar la carácter dinámico de las interacciones DNA proteína en vivo y en regiones específicas del genoma3,4,5. El avance de las tecnologías de secuenciación y la PCR cuantitativa (qPCR) también ha ampliado la capacidad para realizar experimentos de chIP con comparaciones cuantitativas y a través de genomas enteros, lo que es una herramienta poderosa para disecar las interacciones DNA-proteína en múltiples niveles.
Actualmente, la viruta es un método necesario para cualquier grupo de investigación interesado en la regulación de cromatina mediada del genoma como no existen métodos comparables para interrogar directamente el enlace físico entre una histona modificado y un locus genómico específico en vivo. Aunque existen variaciones de este método con secuencia de la generación siguiente a modificaciones de las histonas a lo largo del genoma6,7 , estos enfoques pueden abordar diversas cuestiones científicas y su escala, costo y recursos técnicos pueden ser limitantes para algunos grupos de investigación. Además, chIP-qPCR específica es necesario para complementar estos enfoques proporcionando métodos tanto optimización el protocolo de la viruta antes de iniciar la secuencia y validar los resultados de los conjuntos de datos epigenómica. Espectrometría de masas basado en enfoques para identificar el complemento completo de la histona marcas asociadas con regiones genómicas también han surgido8,9,10,11, sin embargo, estos enfoques tienen algunas limitaciones con respecto a qué regiones del genoma pueden ser sondeadas y requieren conocimientos técnicos y la instrumentación que no estará disponible para todos los grupos de investigación. Por lo tanto, el chIP sigue siendo un método fundamental para el análisis de la abundancia y la distribución de modificaciones de las histonas bajo diversas condiciones para todos los grupos de investigación interesados en epigenética, la cromatina y la regulación de funciones genómicas.
Aquí, describimos un método para modelo de chIP con la levadura de florecimiento Saccharomyces cerevisiae (S. cerevisiae) para investigar la distribución de PTMs histonas en la cromatina. Este enfoque se basa en una serie de componentes de la base de protocolos chIP desarrollado en la levadura y también se aplica a modelo diversos sistemas12,13. Las interacciones entre las histonas modificadas y ADN en la célula se conservan por entrecruzamiento con formaldehído. Tras preparación lisada, fragmentos de cromatina son solubilizados en fragmentos de tamaño uniformemente por digestión con nucleasa micrococcal. Inmunoprecipitación de las histonas modificadas se realiza con anticuerpos comerciales o generados por el laboratorio y cualquier ADN asociado es aislado y analizado para el enriquecimiento en determinadas regiones genomic usando qPCR (figura 1). Para muchas modificaciones de las histonas, la cantidad de ADN obtenido de este protocolo es suficiente para probar más de 25 diferentes loci genómicos por qPCR.
Este método de chIP es muy versátil para el control de la distribución de una modificación de las histonas solo a través de múltiples cepas mutantes o las condiciones ambientales, o para probar múltiples modificaciones de las histonas en las células de tipo salvaje en un número de loci genómicos. Además, numerosos componentes del protocolo son fácilmente ajustables para optimizar la detección de cualquiera de los dos altamente o humildes-abundantes marcas de histona. Finalmente, realizar chIP de histonas modificadas en levadura de florecimiento ofrece la oportunidad de utilizar los controles claves para especificidad de anticuerpos que no están ampliamente disponibles en otros sistemas. Es decir, se pueden generar cepas de levadura que llevan las mutaciones de punto en los residuos de las histonas que son objeto de modificación y, en algunos casos, existe solamente una sola enzima que cataliza un residuo particular histona modificación (ej. lisina de las histonas metiltransferasas). Por lo tanto, chIP puede realizarse en cualquiera de los dos las histona mutante o enzima eliminación cepas analizar la medida en que Unión inespecífica del anticuerpo se puede que ocurre y generar falsos positivos. Este control es particularmente valioso para los anticuerpos del recién desarrollado y puede incluso ser utilizado para validar la especificidad del anticuerpo por modificaciones de las histonas conservados antes de su uso en otros sistemas. Este enfoque complementa otros métodos para probar la especificidad del anticuerpo que distinguen entre Estados de modificación diferentes (por ejemplo, mono-, di - y tri-metilación), incluyendo matrices de péptidos modificados de sondeo y realizar western blot de las histonas o nucleosomas con modificaciones definidas. En general, chIP en levadura de florecimiento es un método eficaz para evaluar la dinámica de la histona MPa por todo el genoma y los mecanismos que regulan su regulación de disección.

<table border="0" cellpadding="0" cellspacing="0" width="876">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast Extract</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">Y20025-1000.0</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Peptone</td>
			<td style="width:301px;">Research Products International (RPI)</td>
			<td style="width:164px;">P20250-1000.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Dextrose</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP350-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycine</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>AC12007-0050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Tris</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">0497-5KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758-500G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">NaCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">BP358-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-Nonylphenyl-polyethylene glycol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">74385</td>
			<td>Equivalent to NP-40</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">MgCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">CaCl2</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">20899-25G-F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">LiCl</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">AC413271000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Dodecyl Sulfate</td>
			<td style="width:301px;">Amresco</td>
			<td style="width:164px;">M107-1KG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Deoxycholate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">30970-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Sodium Acetate</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">S2889</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">NaHCO3</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">S25533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">PMSF</td>
			<td>Sigma-Aldrich</td>
			<td>P7626-5G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Yeast protease inhibitor cocktail</td>
			<td>VWR</td>
			<td>10190-076</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">25 Phenol:24 Chloroform:1 Isoamyl Alcohol</td>
			<td style="width:301px;">VWR Life Science</td>
			<td>97064-824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Ethanol</td>
			<td style="width:301px;">Sigma-Aldrich</td>
			<td style="width:164px;">E7023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Nuclease-Free Water</td>
			<td style="width:301px;">VWR</td>
			<td style="width:164px;">100720-992</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Micrococcal Nuclease</td>
			<td>Worthington Biochemical</td>
			<td>LS004797</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Glycogen</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">R0561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Proteinase K</td>
			<td>Research Products International (RPI)</td>
			<td>P50220-0.1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">RNase A&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>R6513-50MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Bradford Assay Reagent</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">23238</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;BSA Standard 2 mg/mL</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>23210</td>
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			<td height="20" style="height:20px;width:244px;">&#945; H4</td>
			<td>EMD Millipore</td>
			<td>04-858</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H4K16ac</td>
			<td>EMD Millipore</td>
			<td>ABE532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3</td>
			<td style="width:301px;">Abcam</td>
			<td style="width:164px;">ab1791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#945; H3K4me2</td>
			<td>Active Motif</td>
			<td>39142</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;High Rox qPCR Mix</td>
			<td>Accuris qMax Green, Low Rox qPCR Mix</td>
			<td>ACC-PR2000-L-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Protein A/G Magnetic Beads</td>
			<td style="width:301px;">ThermoScientific</td>
			<td>88803</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">magnetic stand for 1.5mL tubes</td>
			<td>Fisher Scientific</td>
			<td>PI-21359</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Acid-Washed Glass Beads</td>
			<td>Sigma-Aldrich</td>
			<td style="width:164px;">G8772</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Microtube Homogenizer</td>
			<td style="width:301px;">Benchmark</td>
			<td>D1030</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">2.0 mL screw-cap tubes with sealing rings</td>
			<td>Sigma-Aldrich</td>
			<td>Z763837-1000EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Gel loading tips</td>
			<td>Fisher Scientific</td>
			<td>07-200-288</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Cuvettes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">50-476-476</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Parafilm</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">50 mL conical tubes</td>
			<td style="width:301px;">Fisher Scientific</td>
			<td style="width:164px;">14-432-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">384-Well PCR Plate</td>
			<td>Fisher Scientific</td>
			<td>AB-1384W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">&#160;Gyratory Floor Shaker</td>
			<td style="width:301px;">New Brunswick Scientific</td>
			<td style="width:164px;">Model G10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Spectrophotometer</td>
			<td style="width:301px;">ThermoScientific</td>
			<td style="width:164px;">ND-2000c</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:244px;">&#160;Real-Time PCR Detection System</td>
			<td style="width:301px;">Bio-Rad</td>
			<td style="width:164px;">1855485</td>
			<td></td>
		</tr>
	</tbody>
</table>

chromatin immunoprecipitation (chip) of histone modifications from saccharomyces cerevisiae

Modificaciones post-traduccionales de las histonas (PTMs), como acetilación, metilación y fosforilación, dinámicamente son reguladas por una serie de enzimas que añadir o quitar estas marcas en respuesta a las señales recibidas por la célula. Estos MPa es principales contribuyentes a la regulación de procesos tales como control de expresión del gene y de reparación del ADN. Inmunoprecipitación de cromatina (chIP) ha sido un acercamiento instrumental para disección de la abundancia y la localización de muchos PTMs histonas por todo el genoma en respuesta a las diversas perturbaciones a la celda. Aquí, se describe un método versátil para la realización de chIP de modificación postraduccional histonas de la levadura de florecimiento Saccharomyces cerevisiae (S. cerevisiae) . Este método se basa en el entrecruzamiento de las proteínas y el ADN mediante tratamiento de formaldehído de las culturas de levadura, generación de lisados de levadura por cordón latiendo, solubilización de fragmentos de cromatina por nucleasa micrococcal e inmunoprecipitación de ADN histonas complejos. ADN asociado con la marca de la histona de interés es purificado y sometido a análisis PCR cuantitativo para evaluar su enriquecimiento en múltiples loci por todo el genoma. Experimentos representativos sondeo la localización de las marcas de histonas H3K4me2 y H4K16ac en wildtype y levadura mutada se discuten para demostrar la interpretación y análisis de datos. Este método es conveniente para una variedad de la histona PTMs y puede realizarse con diferentes cepas mutantes o en presencia de diversas presiones ambientales, lo que es una excelente herramienta para investigar cambios en la dinámica de la cromatina en condiciones diferentes.

Un componente clave de este protocolo es optimizar la concentración de nucleasa micrococcal (MNase) utilizada para digerir la cromatina en fragmentos solubles, como se indica en el paso 10. Esto es fundamental para la obtención de datos de alta resolución con respecto a la distribución de modificaciones de las histonas en regiones genómicas de interés. Debe realizarse una valoración de MNase para determinar la concentración más adecuada para lograr principalmente mono-nucleosomas...

Watch this Scientific Journal Video about Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae at JoVE.com

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Inmunoprecipitación de cromatina (ChIP) de modificaciones de las histonas de Saccharomyces cerevisiae

Aquí, describimos un protocolo para la inmunoprecipitación de cromatina de histonas modificadas de la levadura de florecimiento Saccharomyces cerevisiae. Immunoprecipitated ADN se utiliza posteriormente para PCR cuantitativa para interrogar la abundancia y localización de modificaciones post-traduccionales de las histonas por todo el genoma.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.

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Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

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