Name: preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient
Uploaded: 2014-09-03T15:00:00
Description: Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

Følgende protokoll er i samsvar med retningslinjene i den kanadiske Council of Animal Care og er godkjent av Det medisinske fakultet og Pharmacy Animal Care Utvalget ved Universitetet i Toronto. 1. Utarbeide nødvendig reagenser som beskrevet i Tabell 1. <ol><li> Legg protease og fosfatase (om nødvendig) inhibitorer for alle løsningene sukrose, buffere og DDH 2 O i konsentrasjoner som er skissert i Tabell 1. Viktig utføre alle trinnene ved 4 ° C og pre-kul ...

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Det er flere trinn i prosedyren som er kritiske for et vellykket resultat. I trinn 3, er det viktig at en ensartet grad av homogenisering er oppnådd for hver prøve. Når homogenisere vevet med motordrevet homogenisator, blir en konstant hastighet brukes ikke bare for rotasjon av stampe, men også med det antall slag. Inkubasjonstiden løpet av osmotisk sjokk bør være nøyaktig, siden utvidet homogenisering eller inkubering i hypoton løsning vil lyserer mitokondrier og forurense SPM prøven. <p class="jove_conte...

Neuroner kommuniserer gjennom synapser, og kvaliteten av denne kommunikasjonen er regulert i stor grad ved endringer i sammensetningen av proteiner i synapsen. Spesielt proteiner som ligger i den postsynaptiske tetthet delta i neuronal kommunikasjon ved intimt stillasnevrotransmitterreseptorer med sine signaltransduksjonsveiene systemer en. Videre er varige endringer i styrken av synaptisk effektivitet kontrollert ved tilsetning eller fjerning av reseptorer på postsynaptiske tetthet 1-6. Derfor er isolering og kvantifisering av synaptiske proteiner en nødvendig og nyttig teknikk for å få innsikt i de måter som nevronene reagerer på stimuli og endre synaptiske effekt 7. Denne artikkelen beskriver en vanlig teknikk for å isolere synaptiske proteiner fra gnagere hjernevev etter ultracentrifugation på diskontinuerlige sakkarosegradienter. Den synaptiske plasmamembran brøkdel kan bli beriket og isolert basert pådens tetthet på sukrose, som er empirisk bestemt til å være lik 1,2 M sukrose. Avhengig av den biologiske spørsmålet kan subcellulære fraksjoner separeres ved kontinuerlige eller diskontinuerlige gradienter av enten sukrose eller Percoll. Kontinuerlige gradienter tillate separasjon av proteiner i flere fraksjoner; Dette kan være spesielt nyttig for å demonstrere den ko-lokalisering av proteinene i løpet av en gitt fraksjon 8. Imidlertid er fremstillingen av kontinuerlige graderinger mer arbeidskrevende og er ikke nødvendig for mange anvendelser. Diskontinuerlige gradienter er forholdsvis lettere å forberede og kan brukes til å separere proteiner i et par, generelt definerte fraksjoner. Diskontinuerlige gradienter som er sammensatt av tre lag av sucrose øker molaritet har blitt mye brukt for å isolere proteiner forbundet med den synaptiske plasmamembranen (SPM). Dette synaptiske plasmamembran brøkdel kan behandles videre til den postsynaptiske tetthet fraction (PSD) av vaskemiddelbehandling og isolering av vaskemiddel-uløselige fraksjonen. Når denne prosessen ble først beskrevet i 1960 9,10, ble elektronmikroskopi brukes til å demonstrere de organeller og membraner som grovt definerer den synaptiske plasmamembranen og post-synaptiske tetthet fraksjoner 9-14. Disse studiene viste inkludering av pre-og post-synaptiske membraner og synaptiske vesikler i SPM fraksjon; etter at vaskemiddelbehandling primært elektrontette, post-synaptiske tettheter var synlig. I fremgangsmåten, blir en hypotonisk sjokk som brukes til å klemme ut de synaptiske prosesser fra cellelegemet 10. Dette trinnet tar fordel av det faktum at mitokondriene er mer motstandsdyktig overfor osmotisk sjokk og forbli intakt, og slik at de sedimentere på bunnen av sukrose gradient (figur 1). Ved hjelp av denne samme berikelse teknikk, var de SPM og PSD fraksjoner første biokjemiskdefinert ved polyakrylamidgel-elektroforese, og sekvensering av de store protein-komponenter 15-17. Deretter western blot-analyse ble brukt for å påvise og kvantifisere nivåene av synaptiske proteiner, og videre definere disse fraksjoner (figur 2). Vi har brukt denne teknikken i våre laboratorier for å kvantifisere endringer i de synaptiske nivåer av dopamin transporter som oppstår når Slc6a3 locus er duplisert i mus 18. Vi har også brukt denne teknikken i NMDA reseptor mangelfull mus å avdekke synapse spesifikke reduksjoner i proteiner som er en del av DISC1 interactome 19. Det er tydelig fra western blot analyse at SPM fraksjoner inneholder synaptiske vesikkel membranproteiner, endosome markører, mitokondrie protein, membran assosiert syntetiske enzymer og signaltransduksjon molekyler, så vel som integrerte komponenter av den postsynaptiske tetthet og synaptiske plasmamembraner 20-23. Even PSD fraksjoner kan ha forurensning med rikelig mitokondrie protein, og det kan være nødvendig å utføre en andre gradient sedimentasjon eller flere rensetrinn for å fjerne dem 13. Nylig er kvantitative massespektrometri ga en liste over 100 proteinene i den postsynaptiske densitet alene, så vel som en indikasjon på den relative overflod av disse komponentene 24,25.

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			<td style="width:111px;">LEU001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Pepstatin</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">PEP605.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Benzamidine</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">BEN601.25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium fluoride</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SFL001.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sodium pyrophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SPP310.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:261px;">Sodium orthovanadate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">SOV664.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">&#946;-glycerophosphate</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">GYP001.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Triton X-100</td>
			<td style="width:129px;">BioShop</td>
			<td style="width:111px;">TRX506.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">18G x 1 &#189;&#8221; needle</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">305196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;">1cc syringe</td>
			<td style="width:129px;">BD</td>
			<td style="width:111px;">309659</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:261px;"></td>
			<td style="width:129px;"></td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name of Equipment</td>
			<td style="width:129px;">Company</td>
			<td>Catalog number</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:261px;">Glass Teflon homogenizer Kontes Duall 23</td>
			<td style="width:129px;">VWR</td>
			<td style="width:111px;">KT885450-0023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">IKA Model RW 16 Basic Stirrer</td>
			<td style="width:129px;">IKA Works</td>
			<td style="width:111px;">2572100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall SM-24 Fixed Angle Rotor</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">29017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:129px;">ThermoScientific</td>
			<td style="width:111px;">46910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thinwall Polyallomer Tubes (13.2mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">SW 41 Ti Rotor Swinging Bucket</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">331362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Beckman L-80 Floor Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thickwall Polycarbonate Tubes (3.5mL)</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349622</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TLA-100.3 Fixed Angle Rotor</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;">349481</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:261px;">Beckman TL-100 Tabletop Ultracentrifuge</td>
			<td style="width:129px;">Beckman Coulter</td>
			<td style="width:111px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

Neuronal subcellulære fraksjoneringsteknikker tillater kvantifisering av proteiner som er trafikkert til og fra synapse. Som opprinnelig ble beskrevet i slutten av 1960-tallet, kan proteiner forbundet med den synaptiske plasmamembranen bli isolert ved ultrasentrifugering i en sukrose-densitets-gradient. Når synaptiske membraner er isolert, kan det makromolekylære kompleks kjent som post-synaptiske densitet deretter isolert på grunn av sin uoppløselighet vaskemiddel. Teknikkene som brukes til å isolere synaptiske plasmamembraner og post-synaptiske tetthet proteiner forblir i hovedsak de samme etter 40 år, og er mye brukt i dagens nevrovitenskap forskning. Denne artikkelen beskriver fraksjonering av proteiner forbundet med den synaptiske plasmamembranen og post-synaptiske tetthet ved hjelp av en diskontinuerlig sukrosegradient. Resulterende proteinpreparater er egnet for western blotting eller 2D DIGE analyse.

Fremstillingen av sukrose tetthetsgradient bør resultere i en klar separering av de tre molare oppløsninger av sukrose (0,8, 1,0, og 1,2 M sukrose). Se figur 3A for et eksempel på gradienten før proteinprøve ble tilsatt. Hvis gradienten er forberedt for mye på forhånd, eller om det er forberedt på en benk overflate med vibrasjoner fra annet utstyr, vil gradient bli kompromittert og riktig separasjon vil ikke oppnås. Hvis et klart skille mellom de tre løsningene ikke er synlig, er det tilrådel...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Denne artikkelen beskriver anrikning av proteiner forbundet med den synaptiske plasmamembranen ved ultrasentrifugering på en diskontinuerlig sukrosegradient. Den etterfølgende fremstilling av post-synaptiske tetthet proteiner er også beskrevet. Proteinpreparater er egnet for western blotting eller 2D DIGE analyse.

Utarbeidelse av Synaptic Plasma Membran og postsynaptiske Density Proteiner Ved hjelp av en Discontinuous sukrosegradient

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Research

JoVE Journal

Neuroscience

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

Kvantitativ analyse av Synaptic vesicle Pool Etterfylling i Cultured lillehjernen Granule Neurons bruker FM Fargestoffer

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

En generell metode for evaluering Inkubasjon av Sukrose Begjær i Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Lateral Diffusjon og eksocytose av membran proteiner i dyrkede Neurons vurdert ved hjelp av Fluorescens Recovery og fluorescens-tap photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Post-embedding immunogold merking av Synaptic Proteiner i hippocampus Slice kulturer

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

Utlede Tid Course of Glutamat Lagersalg med en deconvolution Analyse av astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

Nevromuskulære krysset: Måling Synapse Størrelse, fragmentering og endringer i Synaptic Protein Tetthet Bruke Confocal Fluorescensmikroskopi

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...