Name: analysis of histone antibody specificity with peptide microarrays
Uploaded: 2017-08-01T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Dette arbejde blev delvist støttet af Van Andel Research Institute og et forskningsbidrag fra National Institutes of Health (CA181343) til SBR

1. Installere og køre ArrayNinja <ol><li> Download og installer Oracle Virtual Box fra www.virtualbox.org. </li><li> Download og komprimer ArrayNinja virtuelle maskine (VM) fra http://research.vai.org/Tools/arrayninja. </li><li> Åbn Virtual Box og tilføj ArrayNinja VM ved at klikke på &#39;Maskine&#39;, &#39;Tilføj&#39; og vælg arrayninja.vbox fra den mappe, hvor ArrayNinja VM blev gemt. </li><li> Start ArrayNinja ved at vælge det inde i Virtual Box og klikke på den grønne &quot;start&quot; -pile. </li><li> V...

Antistofens pålidelighed i biomedicinske forskningsapplikationer er afgørende 46 , 47 . Dette gælder især i kromatinbiokemi givet antistoffernes position som nøgleværktøjer til de fleste teknikker, der er udviklet til at karakterisere overfladen og fordelingen af ​​histon-PTM&#39;er. Protokollen præsenterede her detaljer en optimeret pipeline til design, fabrikation og anvendelse af peptidmikroarrays for at analysere histon PTM antistofspecificitet. ...

Genomisk DNA pakkes elegant inde i den eukaryote cellekerne med histonproteiner til dannelse af chromatin. Den gentagne underenhed af kromatin er nukleosomet, som består af 147 basepar af DNA viklet omkring en octamer kerne af histonproteiner - H2A, H2B, H3, og H4 1. Kromatin er bredt organiseret i løst pakket eukromatin og tæt pakket heterochromatin domæner. Graden af ​​chromatin-komprimering regulerer i hvilket omfang proteinindustrien kan få adgang til det underliggende DNA til at udføre grundlæggende DNA-templaterede processer som replikation, transkription og reparation. Nøgleregulatorer for genometilgængelighed i forbindelse med chromatin er PTM&#39;er på de ustrukturerede hale- og kerneområder af histonproteiner 2 , 3 . Histon-PTM&#39;er virker direkte ved at påvirke strukturen af ​​chromatin 4 og indirekte througH rekruttering af læserproteiner og deres tilhørende makromolekylære komplekser, der har kromatin remodeling, enzymatisk og stilladsaktiviteter 5 . Undersøgelser af histon-PTM-funktion i løbet af de sidste to årtier tyder på overvejende, at disse mærker spiller nøglerolle i regulering af celleforebyggelse, organisationsudvikling og sygdomsinitiering / progression. Fueled af fremskridt i massespektrometri-baseret proteomteknologi er der fundet mere end 20 unikke histon-PTM&#39;er på mere end 80 forskellige histonrester 6 . Navnlig forekommer disse modifikationer ofte i kombinationer og i overensstemmelse med &quot;histonkode&quot; -hypotesen, viser talrige undersøgelser, at læserproteiner er målrettet mod diskrete regioner af chromatin ved genkendelse af specifikke kombinationer af histon-PTM&#39;er 7 , 8 , 9 . En vigtig udfordring fremadrettet vil være at tildele funktioner til grSkyldige liste over histon-PTM&#39;er og at bestemme, hvordan specifikke kombinationer af histon-PTM&#39;er orkestrerer de dynamiske funktioner, der er forbundet med chromatin. Antistoffer er lynchpinreagenser til påvisning af histon-PTM&#39;er. Som sådan er mere end 1000 histon-PTM-specifikke antistoffer blevet udviklet kommercielt til anvendelse i kromatinbiokemiforskning. Med den hurtige udvikling af DNA-sekventeringsteknologi med høj gennemstrømning anvendes disse reagenser i vid udstrækning af individuelle efterforskere og omfattende epigenomiske &quot;køreplan&quot; -initiativer ( f.eks . ENCODE og BLUEPRINT) i ChIP-seq (chromatinimmunpræcipitation kombineret med næste generations sekventering ) Rørledninger til at generere højopløselige rumlige kort af histon PTM-fordelingsgenet bredt 10 , 11 . Nylige undersøgelser har imidlertid vist, at specificiteten af ​​histon-PTM-antistoffer kan være meget variabel, og at disse reagenser udviser uegnede<html> Dyrebare egenskaber som off-target-epitopgenkendelse, stærk positiv og negativ indflydelse fra nabo-PTM&#39;er og vanskeligheder med at diskriminere modifikationsordren på en bestemt rest ( fx mono-, di- eller trimethyllysin) 12 , 13 , 14 , 15 , 16 , 17 , 18 . Derfor er streng kvalitetskontrol af histon PTM-specifikke antistofreagenser nødvendigt for nøjagtigt at fortolke data genereret med disse værdifulde reagenser. Microarray-teknologien muliggør samtidig forespørgsel af tusindvis af makromolekylære interaktioner i et gennemgående, reproducerbart og miniaturiseret format. Af denne årsag er der blevet udviklet en række mikroarrayplatforme til analyse af protein-DNA 19 ,&quot;&gt; 20, proteinprotein 21 og proteinpeptid-interaktioner 22. Faktisk er histonpeptidmikroarrays fremkommet som en informativ opdagelsesplatform for chromatin biokemiforskning, der muliggør højprofilering af forfattere, viskelæder og læsere af histon PTM&#39;er 15 , 23, 24, og også til analyse af histon antistofspecificitet 17, 25. Beyond deres anvendelse i kromatin og epigenetik forskning, histon peptidarrays har potentiel anvendelighed som en diagnostisk / prognostisk test for systemisk lupus erythematosus og andre autoimmune sygdomme, hvor anti- Chromatin autoantistoffer er dannet 26 , 27 . Her beskriver vi en integreret rørledning, som vi har udviklet til design, fremstilling og køRying histone peptid microarrays at generere specificitet profiler for antistoffer, der genkender histoner og deres PTMs. Rørledningen understøttes af ArrayNinja, en open source, interaktiv software applikation, som vi for nylig udviklede, som integrerer design- og analysestadierne i microarray eksperimenter 28 . ArrayNinja virker bedst i Google Chrome. Kort fortalt anvendes en robotkontakt-mikroarray printer til at deponere et bibliotek af biotinkonjugerede histonpeptider i definerede positioner på streptavidin-coatede glasmikroskop dias. Arrays kan derefter anvendes i et konkurrencedygtigt og parallelt assayformat for at forhøre antistof-epitopinteraktioner ( Figur 1 ). Peptidbiblioteket består af hundredvis af unikke syntetiske peptider, der indeholder PTM&#39;er (lysinacetylering, lysin / argininmethylering og serin / threoninphosphorylering) alene og i relevante kombinationer, der stort set er afledt af proteomiske datasæt. Metoder til peptidsyntese og validering<html> Er beskrevet andetsteds 23 . Data genereret fra vores igangværende histone PTM antistof screening indsats udnytter denne array platform er arkiveret på en offentlig web ressource, Histone Antibody Specificity Database (www.histoneantibodies.com). Især er histonpeptidmikroarrays fremstillet med variationer af denne protokol også blevet anvendt i vid udstrækning for at karakterisere aktiviteten af ​​histon PTM-læserdomænerne 8 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 og mere for nylig til profilering af histon PTM forfatter og viskelæder 24 . /files/ftp_upload/55912/55912fig1.jpg &quot;/&gt; Figur 1: Tegneserieafbildning af den trinvise procedure til antistof screening på en histonpeptid-mikroarray. Biotinylerede histonpeptider med definerede posttranslationelle modifikationer (røde og blå cirkler) er trykt med biotin-fluorescein på streptavidin-belagt glas. Positive interaktioner visualiseres som rød fluorescens. <a href="http://ecsource.jove.com/files/ftp_upload/55912/55912fig1large.jpg" target="_blank">Klik her for at se en større version af denne figur.</a>

<table border="0" cellpadding="0" cellspacing="0" width="746">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Printing Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">PPB</td>
			<td style="width:281px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">BSA</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">2390</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Streptavidin-coated glass microscope slides</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td>439003-25</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">polypropylene 384 well plate</td>
			<td style="width:107px;">Greiner Bio-one</td>
			<td align="right" style="width:105px;">784201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Biotin-fluorescein</td>
			<td style="width:107px;">Sigma</td>
			<td align="right" style="width:105px;">53608</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Aushon</td>
			<td align="right" style="width:105px;">2470</td>
			<td style="width:281px;">Aushon 2470 Microarray Printer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">contact microarray printer</td>
			<td style="width:107px;">Gene Machines</td>
			<td style="width:105px;">OmniGrid 100</td>
			<td style="width:281px;">OmniGrid Microarray Printer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PBS</td>
			<td style="width:107px;">Invitrogen</td>
			<td align="right" style="width:105px;">14190</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Blocking Buffer</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">SBB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Hydrophobic wax pen</td>
			<td style="width:107px;">Vector Labs</td>
			<td style="width:105px;">H-4000</td>
			<td>ImmEdge Hydrophobic Barrier PAP Pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Silicon Gasket</td>
			<td style="width:107px;">Grace Bio-labs</td>
			<td align="right" style="width:105px;">622511</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Hybridization Vessel</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td align="right">267061</td>
			<td>or similar vessel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21244</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-rabbit)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Fluorescent-dye conjugated secondary antibody</td>
			<td style="width:107px;">Life Technologies</td>
			<td>A-21235</td>
			<td style="width:281px;">Alexa Fluor 647 (anti-mouse)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax Imprinter</td>
			<td style="width:107px;">ArrayIt</td>
			<td>MSI48</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tween-20</td>
			<td style="width:107px;">Omnipure</td>
			<td align="right" style="width:105px;">9490</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microarray Scanner</td>
			<td style="width:107px;">Innopsys</td>
			<td style="width:105px;">InnoScan 1100AL</td>
			<td>or equivalent microarray scanner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">EipTitan Histone Peptide Microarray</td>
			<td style="width:107px;">Epicypher</td>
			<td align="right" style="width:105px;">112001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">AbSurance Pro Histone Peptide Microarray</td>
			<td style="width:107px;">Millipore</td>
			<td align="right" style="width:105px;">16668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">MODified Histone Peptide Array</td>
			<td style="width:107px;">Active Motif</td>
			<td align="right" style="width:105px;">13001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Histone Code Peptide Microarrays</td>
			<td style="width:107px;">JPT</td>
			<td style="width:105px;">His_MA_01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Wax</td>
			<td style="width:107px;">Royal Oak</td>
			<td style="width:105px;">GulfWax</td>
			<td>for wax imprinter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Humidified Microarray Slide Hybridization Chamber</td>
			<td style="width:107px;">VWR</td>
			<td style="width:105px;">97000-284</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">High throughput microscope slide washing chamber</td>
			<td style="width:107px;">ArrayIt</td>
			<td style="width:105px;">HTW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Microscope slide centrifuge</td>
			<td style="width:107px;">VWR</td>
			<td>93000-204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 1</td>
			<td>Abcam</td>
			<td align="right" style="width:105px;">8898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Antibody 2</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:105px;">07-473</td>
			<td></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:255px;">Biotinylated histone peptide</td>
			<td style="width:107px;">EpiCypher</td>
			<td>12-0001</td>
			<td style="width:281px;">Example peptide. Similar peptides with various modifications are available from several commercial sources.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ImageMagick</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://www.imagemagick.org/script/index.php</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">ArrayNinja</td>
			<td style="width:107px;"></td>
			<td style="width:105px;"></td>
			<td>https://rothbartlab.vai.org/tools/</td>
		</tr>
	</tbody>
</table>

analysis of histone antibody specificity with peptide microarrays

Post-translationelle modifikationer (PTM&#39;er) på histonproteiner studeres bredt for deres roller i regulering af kromatinstruktur og genekspression. Masseproduktionen og distributionen af ​​antistoffer, der er specifikke for histon-PTM&#39;er, har i høj grad lettet forskningen på disse mærker. Da histon-PTM-antistoffer er nøglereagenser til mange kromatinbiokemiske applikationer, er streng analyse af antistofspecificitet nødvendig for nøjagtig datatolkning og fortsatte fremskridt på området. Denne protokol beskriver en integreret pipeline til design, fabrikation og anvendelse af peptidmikroarrays til profilering af specificiteten af ​​histonantistoffer. Design og analyse aspekter af denne procedure er lettet af ArrayNinja, en open source og interaktiv softwarepakke, vi for nylig udviklede for at strømline tilpasningen af ​​microarray printformater. Denne rørledning er blevet brugt til at screene et stort antal kommercielt tilgængelige og almindeligt anvendte histon PTM antibodiesS, og data genereret fra disse eksperimenter er frit tilgængelige via en online og udvidende Histone Antibody Specificity Database. Ud over histoner kan den generelle fremgangsmåde beskrevet heri anvendes bredt til analysen af ​​PTM-specifikke antistoffer.

Denne protokol er blevet anvendt til at designe og fremstille en peptid-mikroarrayplatform til analyse af histon-PTM-antistofspecificitet. Arrayet spørger om et bibliotek med mere end 300 unikke peptidfunktioner (20-40 rester i længden), der repræsenterer mange af de kendte kombinationer af PTM&#39;er, der findes på kerne- og varianthistonproteiner 38 . Denne rørledning har været en arbejdshest for screeningen af ​​mange almindeligt anvendte og kommercie...

Watch this Scientific Journal Video about Analysis of Histone Antibody Specificity with Peptide Microarrays at JoVE.com

Analysis of Histone Antibody Specificity with Peptide Microarrays

Analyse af histonantistofspecifikitet med peptidmikroarrays

Dette manuskript beskriver metoder til anvendelse af peptidmikraariteknologi til specificitetsprofilering af antistoffer, der genkender histoner og deres posttranslationelle modifikationer.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

The goal of this video is to describe an integrated pipeline for the design fabrication and use of peptide microarrays for profiling the specificities of histone antibodies. Antibodies that recognize histones and their diverse post-translational modifications are key reagents in chromatin biochemistry research. We rely on these tools to deepen our understanding of histone modification function.Recent studies reveal inconsistencies in histone antibody behavior. As antibody choice is the most critical element in epigenetics experiments, vigorous incomprehensive quality control measures are demanded for these reagents. Here, we demonstrate an integrated pipeline we'd developed for designing, fabricating and analyzing peptide microarrays to generate specificity profiles for antibodies that recognize histones and their post-translational modifications.The pipeline is facilitated by Array Ninja, an open source interactive software application that we developed, which integrates the design and analysis stages of microarray experiments. To begin designing the array slide, under the plan a slide layout heading, select the microarray printer that will be used. Then, type, empty in the load plate field and press enter.Set the spot diameter to 275 micrometers and the spot spacing to 100 micrometers. Fill in the remaining parameters appropriately for the experiment, ensuring that the layout allows for sufficient replicates. At the resulting layout preview, mouse over each unique peptide feature on the slide and enter a feature identifier in the popup text box.Once each unique feature has been labeled, click populate. Name the slide layout, and click commit your plate to save the layout and generate a map at the physical locations of each feature in the source plates. Guided by the source plate map, load one to two microliters of each peptide feature into the designated wells of one or more 384 wells, small volume plates, dilute each feature 10 fold with 1x protein microarray printing buffer, supplemented with 1%bovine serum albumin and five micrograms per microliter fluorescin labeled biotin.Then, centrifuge the plates at 500 x G at room temperature for two minutes. Next, empty the waste container at the microarray printer. Fill the washed solution in humidifier containers with sterile distilled water.Enter the array slide parameters in the microarray printer software. Set the washing procedure to a one second wash with the single immersion, the post washed to redip the pins five times, and the humidity to 60%Then, insert encoated glass slides into the substrate platinums. Load the platinums into the platinum elevator.Insert the source plates into the plate holders and load the holders into the source plate elevator. Run the printing process, then, remove the substrate platinums from the instrument. Block the printed slides with 1x protein microarray blocking buffer for 30 minutes while shaking.Then, wash the slides in room temperature, pH 7.6 phosphate buffered saline for 10 minutes. Repeat the wash with a fresh batch of PBS. Dry the slides by centrifugation for 30 seconds at room temperature.Next, pre-heat a microarray wax imprinter at 85 degrees celsius for 30 minutes, or until the wax has melted. Then, insert a slide printed side down, into the holder and align the holder with the imprinter mold. Bring the mold into contact with the slide and hold for two seconds, then, quickly remove the slide from the holder and inspect the wax borders to verify that the arrays are properly enclosed.Store the partitioned slides at four degrees celsius, in a dry dark area. To begin the hybridization procedure, place a partitioned array slide in a plastic dish and cover the slide with hybridization buffer. Equilibrate the slide for 30 minutes at four degree celsius, while shaking at low speed.Dry the slide by spinning in a microarray slide centrifuge at room temperature. Then, add five microliters of each histone PTM antibody solution to at least two wells of the array and incubate at four degree celsius for one hour. Following incubation, remove the antibody solution and wash the array with cold PBS three times for five minutes each at four degrees celsius.Next, incubate the array in a fluorescent dye-conjugated secondary antibody solution. For 30 minutes at four degrees celsius, in the absence of light. Wash the slide three times with cold PBS as before.Dip the array in room temperature 0.1x PBS to remove excess salt and dry the slide by brief centrifugation at room temperature. Image the slide with a microarray scanner at a resolution of at least, 25 microns. To begin the analysis, use image processing software to merge the single channel files from the microarray scanner.Then, open Array Ninja and select the appropriate microarray printer under the, to quantify images header. Fill in the name of the same slide layout in the load plate dialog box, and press enter. Load the merged image file, and adjust the contrast, brightness and mid point as desired.Fill in the array scanner resolution, adjust the top and side margins to align slide layout grid with the image. To analyze a set of wells as replicates or perform other selective analysis, fill in the well coordinates in the iPin, jPin, and subA fields and press enter. Click to toggle zoom, to display a magnified image of the area under the cursor.Then, click spot seek and wait for the process to finish. Repeat the seek function as needed to center the grid circles on the array spots. Reference spots can be selected by pressing the R key and spot showing debris or widely bearing morphology can be deactivated by pressing the A key while hovering over the spot.Once the background reference spots have been selected, and spots with pore morphology or contamination have been deactivated, click populate, followed by, submit to perform the quantification. A bar graph and table will be displayed in a new page. The binding specificities of two histone PTM antibodies were evaluated using this method.A histone PTM antibody designed to target trimethylated lysine 9 on histone H3 was also found to bind to three other trimethylated lysine groups. A silylation of neighboring lysine residues was observed to positively influence targeting of lysine 9 on H3.A histone PTM antibody targeting trimethylated lysine 4 on histone H3, was found to interact with singlely methylated and dimethylated lysine 4 on H3 as well. Phosphorelation of the neighboring three anine six, was observed to negatively impact binding at lysine four in this assay.This pipeline has been used to screen many of the most widely used, commercially available histone antibodies and data generated from these experiments is freely available through the histone antibody specificity database. Variations to this pipeline have been previously described for the analysis of histone readers, writers and erasers, modifying this platform before utility beyond epigenetics research could be easily for any printable library of interest. Several histone peptide microarray platforms are available commercially for users who do not have access to the specialized equipment needed to synthesize peptides and fabricate arrays.

Research

JoVE Journal

Biochemistry

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Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Cellefri produktion af proteoliposomer til funktionel analyse og antistofudvikling rettet mod membranproteiner

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays

In vitro Karakterisering af histon chaperoner ved hjælp af analytiske, pull-down og chaperoning assays

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Dekryptering af molekylær mekanisme for histonsamling ved DNA-gardinteknik

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...