Name: detection of post-translational modifications on native intact nucleosomes by elisa
Uploaded: 2011-04-26T14:00:00
Duration: 7 min 13 s
Description: Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Development of the NU-ELISA method was supported by NIH grant RO1AG023687.

1. Mammalian Cell Culture 
NU-ELISA can be performed on any mammalian cell type that can be grown in culture. We prefer to prepare moderate to large batches of cells so that nucleosomes can be isolated in sufficient quantity to prepare several identically loaded ELISA plates, thus allowing assays with several different antibodies (Abs). The following culture scales provide ample material:
For mouse embyronic stem cells, grow one or two 15 cm plates of cells without feeder cells.
For fibroblasts, grow 5 to 10 15 cm plates to confluence.
2. Isolation of Nuclei
Note: All the steps are on ice with pre-chilled buffers, except as indicated.
<ol>
	<li>Trypsinize cells with 3 ml trypsin per plate, combine, and add 20 ml of ice-cold PBS/butyrate. Centrifuge at 1000 rpm for 5 min.</li>
 <li>Resuspend the cells in 10 ml PBS/butyrate and centrifuge at 1000 rpm for 5 min.</li>
 <li>Resuspend in 4 ml lysis buffer with protease inhibitors (Sigma-Aldrich P-8340). Dounce homogenize 20 strokes with type B pestle on ice.</li>
 <li>Centrifuge at 2000g for 10 min at 4&deg;C. (The supernatant contains cytoplasm, which is not needed for this protocol, but it can be saved for other uses if desired).</li>
 <li>Resuspend the pellet in 2 ml ice cold wash buffer C (with protease inhibitors). </li>
 <li>Layer the resuspended material over 5 ml 30% sucrose cushion, then centrifuge at 2400g for 5 min in a swinging bucket rotor. Nuclei will migrate through cushion, and debris remains at the interface.</li>
 <li>Remove all liquid volume carefully and resuspend the nuclei in 250 &mu;l ice cold wash buffer C + protease inhibitors.</li>
</ol>
3. Isolation of Nucleosomes by in situ Micrococcal Nuclease (MNase) Digestion
We use a procedure in which chromatin is digested in situ within nuclei by infusing them with MNase, followed by a hypotonic treatment to drive free intact nucleosomes into the supernatant. 
<ol>
	<li>Add 3 &mu;l 0.1 M CaCl2 to the sample. Put in 37&deg;C heat block. Allow to assume 37&deg;C temperature.</li>
 <li>Add 2 units MNase (Micrococcal Nuclease, Sigma-Aldrich, dissolved at 2 units/10&mu;l in MNase buffer), then incubate for at 37&deg;C for 12 min, with frequent mixing using a pipet tip.</li>
 <li>Add 6 &mu;l of 0.5 M Na-EDTA, pH 8.0 to stop the reaction. Put on ice.</li>
 <li>Centrifuge at 2000g for 4 min, discard supernatant. Resuspend the pellet in 300 &mu;l 0.2 mM Na-EDTA. Store on ice for 1 h with occasional gentle pipeting. (These hypotonic conditions liberate free nucleosomes from nuclei).</li>
 <li>Centrifuge at 3000 g for 4 min at 4&deg;C. Save the supernatant, which contains free nucleosomes, on ice. </li>
 <li>Resuspend the pellet again with 300 &mu;l 0.2 mM Na-EDTA. Store on ice for 1 h with occasional gentle pipeting.</li>
 <li>Centrifuge at 3000g for 4min at 4&deg;C. Retain the supernatant and combine with the first supernatant from step 5. The resulting mononucleosome preparations can be aliquoted and stored at -80&deg;C. </li> 
</ol>
Note: The amount of chromatin can be crudely quantitated by measuring absorbance at 260 nm of a 10 &mu;l sample added to 990 &mu;l of water. A260=10 (after adjusting for the 1/100 dilution) corresponds to about 1mg/ml of chromatin. Also, during the above procedure, small samples may be retained and later analyzed for their DNA content to monitor the quality and extent of MNase digestions, which should predominantly contain DNA of 146 bp in length. Laddering is indicative of incomplete MNase digestion.
Note: Fig.1 contains a diagrammed summary of nuclear isolation and MNase digestions steps. 
4. Nucleosome-ELISA (NU-ELISA)
We detect PTMs on fractions containing native intact nuclesosomes that have been immobilized on 96 well ELISA microtiter plates. For each sample, we make a series of 2-fold dilutions, and these are coated onto wells in triplicate. 
<ol>
	<li>Coat MaxiSorp plates overnight at 4&deg;C with 50 &mu;l/well of nucleosomes diluted in coating buffer. Suggested serial twofold dilutions of nucleosomes are prepared from top row to the bottom row by adding 0.1 &mu;g, 0.05 &mu;g, 0.025 &mu;g, 0.0125 &mu;g, 0.00625 &mu;g, 0.00313 &mu;g, 0.00156 &mu;g, with coating buffer only (0 &mu;g chromatin) in the bottom row. (Note: plate covers are needed for this step.)</li>
 <li>In the morning, wash the plates 4 times with 200 &mu;l/well PBS/0.5% Tween-20 at room temperature (RT) for a combined total of 10 min with a plate washer. </li>
 <li>Block 1 hr at RT with 100 &mu;l/well PBS/0.05% Tween-20/5% BSA.</li>
 <li>Remove the blocking buffer by shaking the inverted plate briskly over a sink. Cover and store the plates at -20&deg;C, or go directly to the next step.</li>
 <li>Add 1&deg; Abs in a volume of 50 &mu;l/well diluted 1:1000 (or as needed) in PBS/0.05% Tween-20/5% BSA, incubate at RT for 1 hour on a rotator.</li>
 <li>Wash plates 4 times with 200 &mu;l/well PBS/0.5% Tween-20 at RT and for a combined total of 10 min in a plate washer.</li>
 <li>Add horse radish peroxidase (HRP)-conjugated 2&deg; Abs, diluted 1:5000 (or as needed) in PBS/0.05% Tween-20/5% BSA at RT for an hour on a rotator.</li>
 <li>Wash plates 4 times with 200 &mu;l/well PBS/0.5% Tween-20. Each wash is at RT and for a total of 10 minutes with a plate washer.</li>
 <li>Develop the plates by adding 50 &mu;l of TMB substrate to each well for 10 min at RT. Stop the reaction by adding 50 &mu;l/well of 2N H2SO4 and read the plates 450 nm. (Tip: Centrifuge at 1500 rpm for 2 min using a plate rotor to dissipate any air bubbles in the wells prior to reading absorbances). </li>
 <li>Export the readings to spreadsheet files for quantitative and statistical analyses.</li> 
</ol>
Reagents
<table border="1">
 <tbody>	
 	<tr>
 	<td>PBS/butyrate</td>
 </tr>
 <tr>
 	<td>135 mM NaCl</td>
 </tr>

	<tr>
 	<td>2.5 mM KCl</td>
 </tr>
 <tr>
 	<td>8 mM Na2HPO4</td>
 </tr>
 <tr>
 	<td>1.5 mM KH2PO4</td>
 </tr>
 <tr>
 	<td>10 mM Na-butyrate</td>
 </tr> 
 </tbody>
</table>
<table border="1">
	<tbody>
 	<tr>
 	<td>Lysis Buffer</td>
 </tr>
 <tr>
 	<td>250 mM sucrose</td>
 </tr>
 <tr>
 	<td>10 mM Tris-HCl, pH 7.4</td>
 </tr>
 <tr>
 	<td>10 mM Na-butyrate</td>
 </tr>
 <tr>
 	<td>4 mM MgCl2</td>
 </tr>
 <tr>
 	<td>0.1% Triton X-100</td>
 </tr>
 </tbody>
</table>


<table border="1">
	<tbody>
 	<tr>
 	<td>Wash buffer C</td>
 </tr>
 <tr>
 	<td>250 mM sucrose</td>
 </tr>
 <tr>
 	<td>10 mM Tris-HCl, pH 7.4</td>
 </tr>
 <tr>
 	<td>10 mM Na-butyrate</td>
 </tr>
 <tr>
 	<td>4 mM MgCl2</td>
 </tr>
 
 </tbody>
</table>
<table border="1">
	<tbody>
 	<tr>
 	<td>Sucrose Cushion</td>
 </tr>
 <tr>
 	<td>30% (w/v) sucrose in wash buffer C</td>
 </tr>
 </tbody>
</table>
<table border="1">
	<tbody>
 <tr>
 	<td>Microccocal Nuclease Buffer</td>
 </tr>
 <tr>
 	<td>5 mM NaPO4 Buffer, pH 7.0 </td>
 </tr>
 <tr>
 	<td>0.025 mM CaCl2</td>
 </tr>
 </tbody>
</table>
<table border="1">
	<tbody>

 <tr>
 	<td>Coating buffer </td>
 </tr>
 <tr>
 	<td>80ml Solution A + 170ml Solution B + 250ml dH2O</td>
 </tr>
 <tr>
 	<td>Solution A: 0.2 M Na2CO3 </td>
 </tr>
 <tr>
 	<td>Solution B: 0.2 M NaHCO3</td>
 </tr>
 </tbody>
</table> 
5. Representative Results
Using the NU-ELISA method, a series of several identical plates are prepared which can be loaded with chromatin prepared in the form of mononucleosomes. We typically prepare series of identically-loaded plates so that each can be probed with different anti-PTM specific antibodies (Abs). We also always prepare a plate that can be probed with an Ab that detects histones without regard to their modification state total chromatin loading control. This control is essential in order to later quantitatively compare PTM content of nucleosomes prepared from different samples. To quantitate the levels of PTMs within a given chromatin sample we correct the raw absorbance values using this approach: First, we subtract any background signal using values obtained from control wells containing no chromatin (background is usually negligible). We then standardize PTM-specific signals to NU-ELISA results obtained from an identically loaded plate that was assayed with an Ab that detects nucleosomes independent of their modification state. (We have used polyclonal Abs specific for histones H2A or H2B for this purpose). We then determine means and variances for each dilution of chromatin, and use data obtained from the linear portion of the ELISA assay. Detailed examples of NU-ELISA mathematical and statistical analyses have been reported previously4 
<img src="/files/ftp_upload/2593/2593fig1.jpg" alt="Figure 1"/> 
Figure 1. Schematic diagam of NU-ELISA steps. A. Mammalian Cells are harvested; B. Nuclei are separated from cells by Dounce homogenization, C. Disrupted cells are loaded on top of a 30% w/v sucrose cushion, D. After centrifugation, nuclei are deposited in the pellet; E, F. Chromatin are digested in situ to predominantly mononucleomes by MNase; G, H, I. Soluble mononucleosomes are extracted by hypotonic treatment and repeated centrifugation of residual nuclear material. J. Mononucleosomes from different samples (labeled samp. 1 and 2 in this case) are coated on microtiter wells in series of identically-loaded plates and interrogated with PTM-specific Abs and PTM-independent Abs to determine total chromatin loading. K. Depiction of differential signal detection levels in two samples that differ in their PTM content, yet have similar overall chromatin content, as judged by anti-H2B immunoreactivity.

<ol>
	<li>Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., Richmond, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+nucleosome+core+particle+at+2.8+A+resolution.">Crystal structure of the nucleosome core particle at 2.8 A resolution.</a> Nature. 389, 251-260 (1997).</li><li>Jenuwein, T., Allis, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+the+histone+code.">Translating the histone code.</a> Science. 293, 1074-1080 (2001).</li><li>Turner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+and+an+epigenetic+code.">Histone acetylation and an epigenetic code.</a> Bioessays. 22, 836-845 (2000).</li><li>Dai, B., Rasmussen, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epiproteomic+signatures+distinguish+embryonic+stem+cells+from+differentiated+cells.">Global epiproteomic signatures distinguish embryonic stem cells from differentiated cells.</a> Stem Cells. 25, 2567-2574 (2007).</li><li>Tanasijevic, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+accumulation+of+epigen+etic+heterogeneity+during+human+ES+cell+culture.">Progressive accumulation of epigen etic heterogeneity during human ES cell culture.</a> Epigenetics. 4, 330-338 (2009).</li><li>Thomas, J. O., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+fractionation+of+chromatin+and+linker+histones.">Isolation and fractionation of chromatin and linker histones.</a> Chromatin: a practical approach. , 12-14 (1998).</li><li>Thorne, A. W., Cary, P. D., Crane-Robinson, C., Gould, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+and+separation+of+core+histones+and+non-histone+chromosomal+proteins.">Extraction and separation of core histones and non-histone chromosomal proteins.</a> Chromatin: a practical approach. , 38-39 (1998).</li></ol>

No conflicts of interest declared.

NU-ELISA provides a method to ascertain the global status of nucleosome PTMs present within a particular cell type. NU-ELISA studies have shown that global nucleosome modification states differ in comparisons of divergent cell types 4. In addition, NU-ELISA PTM profiles change when cells are exposed to epigenetic modulatory agents such as trichostatin-A or when mouse embyronic stem cells are differentiated 4. The method has also been applied successfully to study the chromatin of human ES cells5. It is important to note the NU-ELISA, in its present form, can detect only the composite signature of PTMs present within the sum total of the cellular epigenome. This differs markedly from genome-wide chromatin immunoprecipitation, which can determine the distribution of a specific PTM across specific genetic loci. 

The initial steps of the NU-ELISA procedures are adapted from previous methods to isolate mononucleosomes from mammalian nuclei6,7. These adapted methods provide an expedient way to obtain high-quality intact mononucleosomes from mammalian cells, and the resulting fractions are comprehensive in their chromatin content, but contain a great deal of additional nuclear material. Since specific Abs are used, contaminating non-nucleosomal material is tolerated well in the NU-ELISA assay, unlike mass-spec approaches that require greater purity. However, NU-ELISA is more sensitive and quantitative than western blotting 4, thus providing good alternatives to westerns and mass spec for the detection of nucleosomal PTMs.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments</th>
 </tr>
 <tr>
 	<td>Micrococcal Nucleae</td>
 <td>Sigma</td>
 <td>N3755</td>
 <td>Make aliquots of 2 U/10&mu;l buffer, stored at -20&deg;C</td>
 </tr>
 <tr>
 	<td>Nunc MaxiSorp Microtiter Plates</td>
 <td>Fisher</td>
 <td>12-565-135</td>
 <td>Adhesive plate covers can also be ordered through Fisher</td>
 </tr>
		<tr>
 	<td>Automated Plate Washer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>1-Step TMB-ELISA
substrate
</td>
 <td>Pierce</td>
 <td>34028</td>
 <td>Store at 4&deg;C pre-warm to RT before use or as directed on the specification sheet</td>
 </tr>
 <tr>
 	<td>Microtiter Plate Reader </td>
 <td>Bio-Rad microplate reader (model: 680)</td>
 <td>&nbsp;</td>
 <td>Most colorimetric plate reader models are suitable </td>
 </tr>
 </tbody>
</table>

detection of post-translational modifications on native intact nucleosomes by elisa

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Watch this Scientific Journal Video about Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA at JoVE.com

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

detection-post-translational-modifications-on-native-intact

Research

JoVE Journal

Biology

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...