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 vil gerne anerkende det danske Forskningsråd (FTP og FNU) til finansiering. ERL anerkender støtte fra en ARC DP tilskud. AB anerkender støtte fra ARC Centre of Excellence i Plant Cell Walls tilskud.

1. Tissue Collection &amp; Forberedelse <ol><li> Collect 100 mg frisk vægt af plantevæv (minimum 10 mg tørvægt) i mindst tre eksemplarer for hvert væv af interesse. De følgende trin beskriver fremstillingen af ​​cellevæggen materiale fra vegetative væv. I tilfælde af storage væv, er un-ønskede stivelse enzymatisk fjernet før man går videre med udvinding af cellevægsbestanddele polymerer som tidligere beskrevet 13. </li><li> Homogeniser prøverne til et fint pulver med flydende nitrogen und...

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CoMPP er en hurtig og følsom fremgangsmåde til profilering af glycan sammensætning af hundreder plante-afledte prøver i løbet af få dage. Denne fremgangsmåde supplerer de allerede tilgængelige bakterier eller pattedyr glycan array-platforme til high-throughput screening af kulhydrat interaktioner med glycan-bindingsproteiner, såsom lectiner, receptorer og antistoffer 16. Med en stor mangfoldighed af prober er tilgængelige til påvisning af cellevægs-glycans, er det muligt at opnå oplysninger om gl...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalognummer </td><td> Kommentarer (valgfrit) </td></tr><tr><td> 3 mm Tungsten Carbide perler </td><td> Qiagen </td><td> 69.997 </td><td></td></tr><tr><td> Collection mikrorør (1,2 mm) </td><td> Qiagen </td><td> 19.560 </td><td> 1,5 ml mikrocentrifugerør kan også anvendes </td></tr><tr><td> Qiagen TissueLyser II </td><td> Qiagen </td><td> 85.300 </td><td></td></tr><tr><td> 3 mm glasperler </td><td> Sigma Aldrich </td><td> Z143928 </td><td></td></tr><tr><td> CDTA </td><td> Sigma Aldrich </td><td> 34.588 </td><td></td></tr><tr><td> Cadmiumoxid </td><td> Sigma Aldrich </td><td> 202.894 </td><td></td></tr><tr><td> 1,2-diaminoethan </td><td> Sigma Aldrich </td><td> 03.550 </td><td></td></<html> tr&gt; <tr><td> Nitrocellulosemembran (0,22 um porestørrelse) </td><td> GE-vand &amp; procesteknologier </td><td> EP2HY00010 </td><td> forskellige pore-størrelser membraner er egnede til forskellige pin typer </td></tr><tr><td> Xact II microarrayer robot </td><td> Labnext </td><td> 001A </td><td> Den Xact II robotten var udstyret med en brugerdefineret 20 x 20 cm keramiske plade, som nitrocellulosemembran er fastgjort </td></tr><tr><td> Xtend RM microarray pins </td><td> Labnext </td><td> 0037-350 </td><td> stifter skal være egnet til at spotte på membraner </td></tr><tr><td> 384 brønds mikrotiterplader (polypropylen) </td><td> Greiner </td><td> 781.207 </td><td></td></tr><tr><td> Anti-glycan monoklonale antistoffer </td><td> Plant Probes / CarboSource / Biosupplies </td><td></td><td> Hjemmesider, 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 (helt molekyle) - Peroxidase antistof fremstillet i geder. </td><td> Sigma </td><td> A9037 </td><td> typen af sekundære antistoffer afhænger af det primære antistof anvendes (f.eks rejst i rotte, mus, geder osv.). </td></tr><tr><td> SIGMA FAST 3,3 &#39;-diaminobenzidin tabletter </td><td> Sigma </td><td> D4293 </td><td> den type udvikle reagens afhænger af de sekundære anvendte antistoffer og metoden til detektion (colourmetric eller chemiluminecent). </td></tr><tr><td> Supersignal West Pico kemiluminescerende substrat </td><td> Thermoscientific </td><td> 34.080 </td><td> se ovenfor </td></tr><tr><td> Xplore Image Processing Software </td><td> LabNext </td><td> 008 </td><td> mange software typer med automatiske gridding værktøjer er tilgængeligeat måle pixelværdi på microarray pletter. </td></tr><tr><td> Plant polysaccharider </td><td> Sigma / Megazyme </td><td></td><td></td></tr></tbody></table>

glycan profiling of plant cell wall polymers using microarrays

Plant cellevægge er komplekse matricer af heterogene glycaner, som spiller en vigtig rolle i fysiologi og udvikling af planter og give de råvarer til menneskelige samfund (f.eks træ, papir, tekstil-og biobrændstof industri) 1,2. At forstå biosyntesen og funktionen af ​​disse komponenter er fortsat udfordrende. Cellevæg glycans er kemisk og konformationelt forskelligartede på grund af kompleksiteten af ​​deres byggesten, de glycosylrester. Disse danner bindinger ved flere positioner og afviger i ringstruktur, isomer eller anomere konfiguration, og endvidere substitueret er med et array af ikke-sukkerrester. Glycan sammensætning varierer i forskellige celle-og / eller vævstyper eller sub-domæner af en enkelt celle væg 3. Desuden er deres sammensætning også ændret under udvikling 1, eller som svar på miljømæssige signaler 4. I exces af 2.000 gener har Plantecellekulturer vægge er komplekse matricer af heterogene glycaner blevet forudsagt at være involveret i cellevæg glycan biosyntese og modifikation i Arabidopsis 5. Imidlertid har relativt få af de biosyntetiske gener blevet funktionelt karakteriseret 4,5. Omvendt genetik fremgangsmåder vanskelige, fordi de gener ofte udtrykkes forskelligt, ofte i lave niveauer, mellem celletyper 6. Også, er mutant undersøgelser ofte hindres af gen-redundans eller kompenserende mekanismer, der sikrer passende cellevæg funktion opretholdes 7. Således nye metoder er nødvendige for hurtigt at karakterisere de mange forskellige glycan strukturer og lette funktionelle genomiktilgange at forstå cellevægsbiosyntesen og modifikation. Monoklonale antistoffer (mAbs) 8,9 er dukket op som et vigtigt redskab til at bestemme glycan struktur og fordeling i planter. Disse anerkender distinct epitoper til stede i større klasser af plantecellevægge glycans, herunder pectiner, xyloglucans, xylaner, mannaner, glucaner og Arabinogalactaner. For nylig deres anvendelse er blevet udvidet til store screening eksperimenter for at bestemme den relative forekomst af glycaner i en bred vifte af plante-og vævstyper samtidigt 9,10,11. Her præsenterer vi en microarray-baseret glycan screeningsmetode kaldet Omfattende Microarray Polymer Profiling (CoMPP) (figur 1 &amp; 2) 10,11, som giver flere prøver (100 sek) skal screenes ved hjælp af en miniaturiseret microarray platform med reduceret reagens og prøvevolumener. The Spot signalerne på microarray formelt kan kvantificeres for at give semi-kvantitative data om glycan epitop forekomst. Denne fremgangsmåde er velegnet til at spore glycan ændringer i komplekse biologiske systemer 12 og giver et samlet overblik over cellevæggen præparat især når forudgående kendskab of dette er ikke tilgængelig.

Den relative forekomst af glycaner i seks vævstyper (anther filamenter, pollen, æggestokke, kronblade, bægerblade og stigmatisering) fra Nicotiana alata blomster blev bestemt ved anvendelse CoMPP. Figur 3A viser et repræsentativt microarray, der er probet med mAb JIM5 specifikt for delvist (lav) methylesterified homogalacturonan (HG), en epitop der forekommer på pectinstoffer polysaccharider 14. Den JIM5 epitopen påvises i CDTA ekstrakter af alle blomster væv men er størst i pollen og mindst ...

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 teknik kaldet<strong&gt; C</strong&gt; Omprehensive<strong&gt; M</strong&gt; Icroarray<strong&gt; P</strong&gt; Olymer Profiling (CoMPP) til karakterisering af plantecellevægge glycans er beskrevet. Denne metode kombinerer af monoklonale antistoffer rettet mod definerede glycan-epitoper med en miniature microarray analytisk platform tillader screening af glycan forekomst i en bred vifte af biologiske sammenhænge.

Glycan profilering af plantecellevægge Polymerer med Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

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