Name: glycan profiling of plant cell wall polymers using microarrays
Uploaded: 2012-12-17T13:00:00
Description: Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

IEM ønsker å takke den danske Forskningsrådet (FTP og FNU) for finansiering. ERL erkjenner støtte fra en ARC DP stipend. AB erkjenner støtte fra ARC Centre of Excellence i Plant Cell Walls stipend.

1. Tissue Collection og klargjøring <ol><li> Samle 100 mg frisk vekt plantemateriale (minimum 10 mg tørrvekt) i minst tre eksemplarer for hvert vev av interesse. Følgende trinn beskriver fremstillingen av celleveggen materiale fra vegetative vev. I tilfelle av lagring vev, er un-ettersøkte stivelse enzymatisk fjernes før fortsetter med utvinning av cellevegg polymerer som tidligere beskrevet 13. </li><li> Homogenisere prøvene til et fint pulver med flytende nitrogen ved hjelp av en Qiagen TissueLyser ...

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CoMPP er en rask og følsom metode for å profilere glycan sammensetningen av hundrevis plante-avledet prøver i løpet av noen dager. Denne metoden utfyller de allerede tilgjengelige bakterielle eller mammalske sukkerkjedene rekke plattformer for high-throughput screening av karbohydrat interaksjoner med glycan-bindende proteiner som lektiner, reseptorer, og antistoffer 16. Med et stort mangfold av sonder tilgjengelige for detektering cellevegg glykaner, er det mulig å få informasjon om sukkerkjedene epito...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer (valgfritt) </td></tr><tr><td> 3 mm Tungsten Carbide perler </td><td> Qiagen </td><td> 69997 </td><td></td></tr><tr><td> Samling mikrorør (1,2 mm) </td><td> Qiagen </td><td> 19560 </td><td> 1,5 ml mikrofugerør rør kan også brukes </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85300 </td><td></td></tr><tr><td> 3 mm glassperler </td><td> Sigma Aldrich </td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> Sigma Aldrich </td><td> 34588 </td><td></td></tr><tr><td> Kadmium oksid </td><td> Sigma Aldrich </td><td> 202894 </td><td></td></tr><tr><td> 1,2-diaminoetan </td><td> Sigma Aldrich </td><td> 03550 </td><td></td></<html> tr&gt; <tr><td> Nitrocellulose membran (0,22 mikrometer porestørrelse) </td><td> GE-vann og prosess teknologier </td><td> EP2HY00010 </td><td> forskjellige pore størrelse membraner er egnet for ulike pin typer </td></tr><tr><td> Xact II microarrayer robot </td><td> Labnext </td><td> 001A </td><td> den Xact II robot ble utstyrt med en tilpasset 20 x 20 cm keramisk plate som den nitrocellulose membran er festet </td></tr><tr><td> Xtend RM microarray pins </td><td> Labnext </td><td> 0037-350 </td><td> pinner må være egnet for å fange opp på membraner </td></tr><tr><td> 384 brønner mikrotiterplater (polypropylen) </td><td> Greiner </td><td> 781207 </td><td></td></tr><tr><td> Anti-sukkerkjedene monoklonale antistoffer </td><td> Plant Sonder / CarboSource / Biosupplies </td><td></td><td> Nettsteder, PlantProbes ( <a href="http://www.plantprobes.net" target="_blank">www.plantprobes.net</a> ), Carbosource (ww.carbosource.net &quot;target =&quot; _blank &quot;&gt; www.carbosource.net) og Biosupplies ( <a href="http://www.biosupplies.com.au" target="_blank">www.biosupplies.com.au</a> ). </td></tr><tr><td> Anti-rotte IgG (hele molekylet) - Peroxidase antistoff produsert i geit. </td><td> Sigma </td><td> A9037 </td><td> typen sekundære antistoffer avhenger primære antistoff som brukes (for eksempel opp i rotte, mus, geit etc.). </td></tr><tr><td> SIGMA FAST 3,3 &#39;-Diaminobenzidine tabletter </td><td> Sigma </td><td> D4293 </td><td> typen utvikle reagens avhenger sekundære antistoffer brukes og deteksjonsmetode (colourmetric eller chemiluminecent). </td></tr><tr><td> SuperSignal West Pico kjemiluminescerende Substrat </td><td> Thermoscientific </td><td> 34080 </td><td> se ovenfor </td></tr><tr><td> Xplore bildebehandling </td><td> LabNext </td><td> 008 </td><td> mange programvare typer med automatiske kloss verktøy er tilgjengeligå måle piksel verdien av microarray flekker. </td></tr><tr><td> Plant polysakkarider </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Plant celleveggene er komplekse matriser av heterogene glykaner som spiller en viktig rolle i fysiologi og utvikling av planter og gi råstoff for menneskelige samfunn (for eksempel tre, papir, tekstiler og biobrensel industri) 1,2. Men, forstå biosyntesen og funksjon av disse komponentene forblir utfordrende. Cellevegg glykaner er kjemisk og conformationally mangfoldig på grunn av kompleksiteten i deres byggeklosser, de glykosyl rester. Disse danner bindinger ved flere posisjoner og skiller seg i ringstruktur, isomerisk eller anomere konfigurasjon, og i tillegg er substituert med en rekke ikke-sukkerrester. Glycan sammensetningen varierer i forskjellige celle-og / eller vevstyper eller selv sub-domener av en enkelt celle vegg 3. Videre er deres sammensetning også modifisert under utviklingen 1, eller i respons til miljømessige signaler 4. I exsessen av 2000 gener har er Plant celleveggene komplekse matriser av heterogene glykaner blitt spådd å være involvert i celleveggen glycan biosyntese og modifikasjon i Arabidopsis 5. Imidlertid har relativt få av biosyntetiske gener blitt funksjonelt karakterisert 4,5. Omvendt genetikk tilnærminger er vanskelig fordi genene er ofte forskjellig uttrykk, ofte på lave nivåer, mellom celletyper 6. Dessuten er mutant studier ofte hindret av genet redundans eller kompenserende mekanismer for å sikre hensiktsmessig celleveggen funksjon opprettholdes 7. Dermed nye tilnærminger for å raskt karakterisere det varierte utvalget av sukkerkjedene strukturer og legge til rette funksjonell genomikk tilnærminger til forståelse celleveggen biosyntese og modifikasjon. Monoklonale antistoffer (mAbs) 8,9 har dukket opp som et viktig verktøy for å bestemme glycan struktur og distribusjon i planter. Disse gjenkjenner distinct epitoper til stede innenfor store klasser av plante cellevegg glykaner, inkludert pectins, xyloglucans, xylans, mannans, glukaner og arabinogalactans. Nylig bruken har blitt utvidet til store screening eksperimenter for å bestemme den relative overflod av glykaner i et bredt spekter av anlegg og vevstyper samtidig 9,10,11. Her presenterer vi en microarray-baserte glycan screening metode som kalles Omfattende Microarray Polymer Profiling (CoMPP) (figur 1 og 2) 10,11 som gjør at flere prøver (100 sek) skal skjermes ved hjelp av en miniatyrisert microarray plattform med redusert reagens og prøvevolumer. Spot signaler på microarray kan bli formelt kvantifisert å gi semi-kvantitative data om glycan epitop forekomst. Denne tilnærmingen er godt egnet til å spore endringer i sukkerkjedene komplekse biologiske systemer 12 og gir en global oversikt over celleveggen preparat spesielt når forkunnskaper of dette er utilgjengelig.

Den relative overflod av glykaner i seks vevstyper (anther filamenter, pollen, eggstokker, kronblad, begerblad og stigma) fra Nicotiana Alata blomster ble fastsatt ved hjelp CoMPP. Fig. 3A viser et representativt microarray som har blitt analysert med mAb JIM5 spesifikk for delvis (lav) metylforestringsgrad homogalacturonan (HG), en epitop som forekommer på pektinstoffer polysakkarider 14. Den JIM5 epitop er oppdaget i CDTA ekstrakter av alle blomst vev er imidlertid høyest i pollen og lavest i sti...

Watch this Scientific Journal Video about Glycan Profiling of Plant Cell Wall Polymers using Microarrays at JoVE.com

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

En teknikk som kalles<strong&gt; C</strong&gt; Omprehensive<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strong&gt; Olymer profilering (CoMPP) for karakterisering av plante cellevegg glykaner er beskrevet. Denne metoden kombinerer spesifisitet av monoklonale antistoffer rettet mot definerte glycan-epitoper med en miniatyr microarray analytisk plattform som tillater screening av glycan forekomst i et bredt spekter av biologiske sammenhenger.

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