Name: brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization
Uploaded: 2017-05-12T15:00:00
Description: Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

Dette arbejde blev støttet af Ministerio de Economia y Competitividad / Instituto de Salud Carlos III (SAF2014-55700-P, PCIN-2013-019-C03-03 og PIE14 / 00034), Instituciò Catalana de Recerca i Estudis Avançats (ICREA Academia-2010 ), Og Agentschap voor Innovatie door Wetenschap en Technologie (SBO-140028) til FC Også X. M, VF-D og FC tilhører den akkrediterede forskningsgruppe &quot;Neuropharmacology and Pain&quot; (Generalitat de Catalunya, 2014 SGR 1251) . Arbejdet blev også støttet af &quot;Programa Pesquisador Visitante Especial-Ciência sem Fronteiras&quot; fra CAPES (Brasilien) til FC

Alle dyreforsøgsprocedurer blev godkendt af University of Barcelona Committee for Animal Use and Care (CEEA) i overensstemmelse med retningslinjerne beskrevet i Vejledningen om pleje og brug af laboratoriedyr 11 og efter Det Europæiske Fællesskab, lov 86/609 / CCE, FELASA og ARRIVE retningslinjer. Mus er således anbragt i standardburer med ad libitum adgang til mad og vand og opretholdes under kontrollerede standardbetingelser (12 h mørk / lyscyklus startende ved 7:30, 22 ° C og 66...

<ol>
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The protocol presented here constitutes a powerful biochemical tool for the study of the subsynaptic distribution of specific proteins within any brain region. However, there are some drawbacks inherent to the technique that deserve to be highlighted here. For instance, one of the main limitations is the relatively large amount of tissue needed to purify a reasonable amount of protein in order to perform the immunoblot analysis of all subsynaptic fractions. This issue might be related to the fact that synapses (i.e.,...

Synaptisk transmission er afhængig af synaps fysiske integritet, et koncept, der var planlagt allerede i 1897 af Foster and Sherrington 1 . Således er forståelse for fordelingen af ​​centrale neurotransmissionskomponenter ( f.eks. Ionkanaler, receptorer, etc. ) afgørende for at belyse synaptisk funktion både under normale og patologiske forhold. Elektronmikroskopi (EM) har bidraget enormt til den nuværende ultrastrukturelle opfattelse af prototypiske centralnervesystems (CNS) synaps. EM har således på en sådan måde fastslået forskellene mellem præ- og postsynaptiske tætheder, som adskilles af en spalt af en ret ensartet afstand (~ 25 nm) 2 . Interessant nok udviser det postsynaptiske apparat en relativt kontinuerlig, elektron-tæt fortykning under sin plasmamembran, den såkaldte postsynaptiske densitet eller PSD 2 . Omvendt, ved det præsynaptiske apparat, en notifikationsblankEt diskontinuerligt cytomatrix netværk er anbragt lige under plasmamembranen, hvilket er afgørende for tilpasningen og dockningen af ​​synaptiske vesikler til den plasmamembranaktive zone 3 . Derfor udgør EM den gyldne eksperimentelle tilgang til undersøgelse af fordelingen af ​​proteiner inden for strukturelt bevarede CNS-synapser. Imidlertid er informationen fra elektronmikrografier statisk. Faktisk viser akkumulerende bevismaterialer, at in vivo synapser er ekstremt dynamiske og oplever således dramatiske strukturelle ændringer ved vedvarende synaptisk transmission. Desuden kan morfologien og sammensætningen af ​​synapser ændre sig gennem forskellige CNS-regioner og ved udvikling, modning, aldring og udvikling af neuropatologiske tilstande. Samlet set repræsenterer en protokol om isolering af proteiner, der tilhører forskellige synaptiske rum i fysiologiske forhold, et værdifuldt redskab til en mere omfattende undersøgelse af synaptisk funktion. <p cLass = &quot;jove_content&quot;&gt; Her beskriver vi denne form for komplementær eksperimentel tilgang, som giver mulighed for den præparative biokemiske berigelse af de forskellige synaptiske membrankamre, nemlig ekstra-, præ- og postsynaptiske membranområder. Denne membranfraktioneringsmetode, først beskrevet af Philips et al . (2001) 4 , er baseret på et pH-skifte, som svækker de klæbende interaktioner der forekommer inden for det præ- og postsynaptiske apparat. For det første er det muligt at skelne adhærensforbindelsen, der indeholder det præ- og postsynaptiske apparat, og som opretholdes fra det ekstrasynaptiske membrandomæne, som er solubiliseret og således kan ekstraheres fra de synaptiske kontakter, ved at anvende milde vaskemidler ved pH 6,0. Efterfølgende hæver pH-værdien fra 6,0 til 8,0 i nærvær af milde vaskemidler styrken af ​​adherensforbindelsen, som holder den presynaptiske aktive zone tæt bundet til den postsynaptiske densitet. Derfor er det presynaptiske rum såledesLubilized og kan adskilles fra den postsynaptiske tæthed, som hovedsagelig bevares, fordi den anvendte koncentration af detergent ikke fremmer dets solubilisering 4 . Interessant nok kan fraktioneringseffektiviteten, som til sidst er højere end 90%, bekræftes af forskellige subsynaptiske markører: i ) synaptosomalt associeret protein 25 (SNAP-25) fra den presynaptiske aktive zone; Ii ) Synaptophysin, fra den ekstrasynaptiske fraktion ( dvs. uden for den aktive zone og inklusiv mikrosomer); Og iii ) postsynaptisk densitetsprotein 95 (PSD-95), fra den postsynaptiske densitet. Især er denne hjernemembranfraktioneringsmetode blevet anvendt med succes. Følgelig har det været muligt præcist at bestemme den subsynaptiske lokalisering af forskellige receptorer, såsom alfa-amino-3-hydroxy-5-methyl-4-isoxazolpropionsyre (AMPA) receptorer 5 , adenosin A1 receptor (A1R) 6 ,Adenosin A 2A- receptor (A 2A R) 7 , adenosintrifosfat (ATP) P2-receptorer 8 , nicotinacetylcholinreceptorunderenheder 9 og Parkinsons sygdomsassocierede receptor GPR37 10 . En række begrænsninger kan dog hæmme den korrekte vurdering af den synaptiske fordeling af et bestemt neuronalt protein. Således beskriver vi i denne procedure ikke kun hele protokollen fuldstændigt, men vi fremhæver også nogle kritiske punkter, der skal overvejes, såsom den ret store mængde væv, der kræves, lavproteinudbyttet og det obligatoriske krav til at validere effektiviteten af Hver adskillelse inden udførelse af det konkrete eksperiment.

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		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sucrose</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,211,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">CaCl2</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">2,112,211,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">MgCl2&#183;6H2O</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,313,961,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Protease inhibitor cocktail Set III</td>
			<td style="width:249px;">Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">535140</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Trizma Base</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tris-HCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,236,541,209</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Triton X-100</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">X100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">SDS</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">L3771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Glycerol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">G5516</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Bromophenol Blue</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,311,651,604</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Dithiothreitol</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">D0632</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Tween 20</td>
			<td style="width:249px;">Sigma, St. Louis, MO, USA</td>
			<td style="width:160px;">P2287</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Non fat dry milk</td>
			<td style="width:249px;"></td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">NaCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,216,591,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KCl</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,314,941,210</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">KH2PO4</td>
			<td style="width:249px;">Merck</td>
			<td style="width:160px;">4873</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Na2HPO4</td>
			<td style="width:249px;">Pancreac Qu&#237;mica SL, Barcelona, Spain</td>
			<td style="width:160px;">1,316,781,211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Basic 20 pH</td>
			<td style="width:249px;">Crison, Alella, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Polytron VDI 12 Adaptable Homogenizer</td>
			<td style="width:249px;">VWR, Radnor, PA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:128px;">Ultra-Clear Tubes (14x89mm)</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;">344059</td>
			<td style="width:167px;">Tubes should be filled almost completely when used to prevent collapsing due to ultracentrifugation.</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Amicon Ultra-15 Centrifugal filters Ultracel -10K</td>
			<td style="width:249px;">Merck Millipore, Darmstadt, Germany</td>
			<td style="width:160px;">UFC901008</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Centrifuge 5430R</td>
			<td style="width:249px;">Eppendorf, Hamburgo, Germany</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Optima L-90K Ultracentrifuge</td>
			<td style="width:249px;">Beckman Coulter, Hospitalet de Llobregat, Barcelona</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Sonifier 250</td>
			<td style="width:249px;">Branson, Danbury, Connecticut</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Amersham Imager 600</td>
			<td style="width:249px;">GE Healthcare Europe GmbH, Barcelona, Spain</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Disposable Glass Pasteur Pippetes 230 mm</td>
			<td style="width:249px;">VWR, Radnor, PA, USA</td>
			<td style="width:160px;">612-1702</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Compact Balance EK-610</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">Pierce&#8482; BCA Protein Assay Kit</td>
			<td style="width:249px;">Pierce Biotechnology, Rockford, IL, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">SuperSignal west pico chemiluminescent substrate</td>
			<td style="width:249px;">Thermo Fisher Scientific Inc., Waltham, MA, USA</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">GR 200 Precision Balance</td>
			<td style="width:249px;">A&#38;D, Tokyo, Japan</td>
			<td style="width:160px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-GPR37</td>
			<td style="width:249px;">Homemade antibody anti-GPR37 produced and validated in Francisco Ciruela Laboratory.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies used at a final concentration of 0.250ug/ml</td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:128px;">Anti-SNAP-25, anti-PSD-95, anti-synaptophysin</td>
			<td style="width:249px;">Abcam, Cambridge, United Kingdom</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Primary antibodies diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-mouse IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:10000</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:128px;">HRP-conjugated goat anti-rabbit IgG</td>
			<td style="width:249px;">Thermo Fisher Scientific, Waltham, MA, USA.</td>
			<td style="width:160px;"></td>
			<td style="width:167px;">Secondary antibody diluted 1:30000</td>
		</tr>
	</tbody>
</table>

brain membrane fractionation: an ex vivo approach to assess subsynaptic protein localization

Vurdering af den synaptiske proteinsammensætning og funktion udgør en vigtig udfordring inden for neurovidenskab. Det er imidlertid ikke let at vurdere neurotransmission, der forekommer inden for synaps, fordi den er stærkt reguleret af dynamiske protein-proteininteraktioner og fosforyleringshændelser. Når en metode anvendes til at studere synaptisk transmission, er det derfor vigtigt at bevare disse forbigående fysiologiske modifikationer. Her præsenterer vi en hjernemembranfraktioneringsprotokol, der repræsenterer en robust procedure til isolering af proteiner, der tilhører forskellige synaptiske rum. Med andre ord beskriver protokollen en biokemisk metode til udførelse af proteinberigelse fra presynaptiske, postsynaptiske og ekstrasynaptiske rum. For det første opnås synaptosomer eller synaptiske terminaler fra neuroner, som indeholder alle synaptiske rum ved hjælp af en diskontinuerlig sucrosegradient. Det bemærkes, at kvaliteten af ​​dette indledende synaptiske membranpræparat er critical. Derefter opnås isoleringen af ​​de forskellige subsynaptiske rum med let solubilisering under anvendelse af milde detergenter ved forskellige pH-forhold. Dette muliggør adskillelse ved gradient- og isopykniske centrifugeringer. Endelig valideres proteinberigelse i de forskellige subsynaptiske rum ( dvs. præ-, post- og ekstrasynaptiske membranfraktioner) ved hjælp af immunoblot-analyse ved anvendelse af velkarakteriserede synaptiske proteinmarkører ( dvs. SNAP-25, PSD-95 og synaptofysin, Henholdsvis), hvilket muliggør en direkte vurdering af den synaptiske fordeling af et hvilket som helst bestemt neuronalt protein.

Den beskrevne metodik er i vid udstrækning anvendt til den subsynaptiske analyse af neuronale proteiner generelt og til isolering og biokemisk karakterisering af synaptiske receptorer 5 , 6 , 7 , 8 , 9 i særdeleshed. Interessant nok viser det repræsentative resultat, der vises her, brugen af ​​denne eksperimentelle proc...

Watch this Scientific Journal Video about Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization at JoVE.com

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Her præsenterer vi en hjernemembranfraktioneringsprotokol, der repræsenterer en robust procedure til isolering af proteiner, der tilhører forskellige synaptiske rum.

Hjernemembran Fraktionering: An<em&gt; Ex Vivo</em&gt; Tilnærmelse til vurdering af subsynaptisk protein lokalisering

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Research

JoVE Journal

Biochemistry

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Brug Stillads Liposomer at Rekonstituer Lipid-proksimal protein-protein interaktioner<em&gt; In vitro</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Assay for Phosphorylation and Microtubule Binding Along with Localization of Tau Protein in Colorectal Cancer Cells

Assay for fosforylering og mikrotubulus bindende sammen med lokalisering af Tau Protein i kolorektal cancerceller

The microtubule-associated protein tau is a neuronal protein that localizes mostly in axons. Generally tau is essential for normal neuronal functioning ...

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

Påvisning af små GTPase Prenylation og GTP bindende ved hjælp af membran fraktionering og GTPase-forbundet Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

Forberedelse af grønkorn sub rum fra Arabidopsis til analyse af Protein lokalisering af Immunoblotting eller Proteomics

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Et fluorescens udsving spektroskopi Assay af Protein-Protein interaktioner på celle-celle kontakter

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

Evaluering af protein-protein interaktioner ved hjælp af en on-membran fordøjelse teknik

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

Interaktioner med og membran Permeabilisering af hjernen mitochondrier af amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

Native Cell Membrane Nanopartikler System for Membran Protein-Protein Interaktion Analyse

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysomprofilering uden gradueringsproducenter eller fraktioneringssystemer

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Vurdering af indsendeokondrial protein lokalisering i spirende gær saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...