Name: enrichment of detergent-insoluble protein aggregates from human postmortem brain
Uploaded: 2017-10-24T12:30:00
Description: Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Forfatterne takke Drs. Jim Lah og Allan Levey, Emory Department of Neurology, for nyttige kommentarer og forslag. Dette arbejde var delvis finansieret af den accelererende medicin partnerskab grant (U01AG046161-02), Emory Alzheimer's Disease Research Center (P50AG025688) og en National Institute on Aging yder (R01AG053960-01) til N.T.S. Denne forskning blev også delvist understøttet af neuropatologiske kernen i Emory neurovidenskab NINDS Core faciliteter grant, P30NS055077.

etik erklæring: alle hjernens væv blev indhentet fra Emory Alzheimers &#39; s sygdom Research Center (ADRC) hjerne Bank. Menneskelige postmortem væv er anskaffet under passende institutionelle Review Board (IRB) protokoller.
 1. homogenisering og fraktionering <ol><li>væv udvalg Bemærk: frosne postmortem frontale cortex væv fra sunde kontrol (Ctl) og patologisk bekræftede AD tilfælde blev udvalgt fra Emory ADRC hjernen bank (n = 2). Post mortem Neurop...

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Heri vi præsentere og diskutere en forkortet trinvis vaskemiddel-fraktionering protokol, der er gældende for en lang række eksperimentelle applikationer lige fra massespektrometri-baseret proteomics analyser til funktionelle protein misfoldning assays og western blotting. 5 , 6 , 7 , 10 denne metode er måske mest effektiv når bruges til at studere neurodegenerative proteinopathies som Alzh...

Neurodegenerative sygdomme såsom Alzheimers sygdom (AD), Parkinsons sygdom (PD), Huntington's chorea (HD), amyotrofisk lateral sklerose (ALS), og de nært beslægtede prionsygdomme er proteinopathies karakteriseret ved gradvis akkumulering af Detergent-uopløselige protein aggregater i hjernen med ledsager kognitiv tilbagegang. 1 , 2 denne delte patologiske funktion menes at spille en central rolle i ætiologi og Patofysiologi af disse neurodegenerative sygdomme. 2 disse aggregater består typisk af polymert amyloid fibre, der består af gentagne enheder af fejlfoldede protein udstiller på tværs af β-struktur. 1 , 2 , 3 , 4 biokemisk, amyloid aggregater er meget modstandsdygtigt over for kemisk eller termisk denaturering og oploesning,3 som giver unikke udfordringer til deres rensning, analyse og studere via traditionelle biokemiske teknikker. 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 ikke overraskende, detergent-uopløselige proteinfraktion har været genstand for megen forskning i Patofysiologi af neurodegenerative sygdomme der involverer ophobning af fejlfoldede proteiner. 6 , 12 , 13 , 14
Biokemiske fraktionering teknikker har ofte været udnyttet til at berige de detergent-uopløselige fraktion fra postmortem hjernen homogeniseret. 6 , 12 , 13 , 14 en af de mest almindelige metoder omfatter den sekventielle udvinding af væv homogeniseret med buffere og rengøringsmidler for at øge stringens, efterfulgt af ultracentrifugering at partitionere de opløselige og uopløselige fraktioner. Et almindeligt anvendte sekventielle fraktionering protokol6,14 indebærer homogenisering af frosne vævsprøver i en buffer, vaskemiddel-gratis lavt saltindhold (LS) og de resulterende uopløselige pellets derefter ekstraheres sekventielt med buffere med højt saltindhold, nonioniske detergenter, high saccharose, som Ioniske detergenter og endelig chaotropes urinstof. 6 , 14 en indlysende godtgørelse af sådanne en sekventiel fraktionering protokoller er betydelig tid og arbejdskraft engagement kræves for at fuldføre dem. Herunder homogenisering og ultracentrifugering, en typisk fem-trins fraktionering protokol kan tage flere timer eller endda dage at fuldføre. 4 , 6 , 7 , 10 , 15 , 16 , 17 , 18 Derudover så mange patologiske protein aggregater forblive uopløseligt i alle, men de hårdeste betingelser19,20 de fleste af de genererede fraktioner er af begrænset værdi. De mindre strenge fraktionering trin udnytter saltkoncentrationer og nonioniske detergenter er således i vid udstrækning redundante.
Tidligere undersøgelser har vist, at den ioniske vaskemiddel N-lauryl-sarcosine (sarkosyl) er en fremragende kandidat til en forenklet trinvis vaskemiddel fraktionering protokol. 5 , 6 , 12 , 13 , 14 , 21 , 22 , 23 som en denaturering vaskemiddel, sarkosyl er strenge nok til at solubilize størstedelen af indbygget foldede proteiner i hjernen uden solubilizing fejlfoldede protein aggregater sammensat af beta-amyloid (Aβ),6,11 fosforyleres tau (pTau),6 TAR DNA-bindende protein 43 (TDP-43),14 alpha-synuclein,12,13 scrapie,23 eller U1 small nuclear ribonucleoproteins (U1 snRNPs) som U1 - 70 K. 5 , 21 , 22 Som sarkosyl er mindre strenge end den allestedsnærværende anioniske detergent sodium dodecyl sulfat (SDS), bevarer det mindre robust oligomere former af fejlfoldede protein aggregater, der ikke kan modstå SDS behandling. 9
Vi beskrev tidligere, en forkortet vaskemiddel-fraktionering-protokol, der opnåede resultater sammenlignes med de mere besværlige sekventielle fraktionering metoder. 5 ved at udelade de mindre strenge fraktionering skridt, vi har kunnet udvikle en letkøbt trinvis fraktionering protokol for berigelse af detergent-uopløselige protein aggregater fra postmortem menneskelige hjerne. 5 denne detaljerede protokollen beskrevet heri er velegnet til en lang række eksperimentelle applikationer lige fra Western blotting og massespektrometri-baseret proteomics funktionelle protein misfoldning og sammenlægning såning-undersøgelser. 5 , 6 , 21

<table>
	<tbody>
		<tr>
			<td>Protease and phosphatase inhibitor cocktail, EDTA-free (100X)</td>
			<td>Thermo Fisher</td>
			<td>78441</td>
			<td>protease &#38; phosphatase inhibitor cocktail</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator System (ultrasonicator)</td>
			<td>Fisher Scientific</td>
			<td>FB505110</td>
			<td>microtip ultrasonicator</td>
		</tr>
		<tr>
			<td>Optimax TLX Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>361545</td>
			<td>refrigerated ultracentrifuge</td>
		</tr>
		<tr>
			<td>TLA120.1 rotor</td>
			<td>Beckman Coulter</td>
			<td>362224</td>
			<td>ultracentrifuge rotor</td>
		</tr>
		<tr>
			<td>500 ul (8x34mm) polycarbonate tubes, thickwall</td>
			<td>Beckman Coulter</td>
			<td>343776</td>
			<td>ultracentrifuge tubes for TLA120.1 rotor</td>
		</tr>
		<tr>
			<td>4X SDS sample buffer</td>
			<td>Home-made</td>
			<td>N/A</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>TCEP solution, neutral pH</td>
			<td>Thermo Fisher</td>
			<td>77720</td>
			<td>reducing agent</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer</td>
			<td>Thermo Fisher</td>
			<td>37542</td>
			<td>blocking buffer</td>
		</tr>
		<tr>
			<td>(TBS) blocking buffer + 0.05% Tween 20</td>
			<td>Thermo Fisher</td>
			<td>37543</td>
			<td>blocking buffer and antibody diluent</td>
		</tr>
		<tr>
			<td>4-12% Bolt Bis-Tris Plus gels, 10-well</td>
			<td>Thermo Fisher</td>
			<td>NW04120BOX</td>
			<td>precast SDS-PAGE gels</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher</td>
			<td>B0002</td>
			<td>SDS-PAGE running buffer</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt (sarkosyl)</td>
			<td>Sigma Aldrich</td>
			<td>L5777-50G</td>
			<td>detergent</td>
		</tr>
		<tr>
			<td>1.5 ml polypropylene Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749521-1500</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>cordless motor for Pellet Pestle</td>
			<td>Kimble Chase</td>
			<td>749540-0000</td>
			<td>homogenization tool</td>
		</tr>
		<tr>
			<td>Anti-Tau-2 (pan tau) antibody</td>
			<td>Chemicon</td>
			<td>MAB375</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-threonine 231 Tau antibody</td>
			<td>Millipore</td>
			<td>MAB5450</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-phospho-seroine 202 and threonine 205 Tau antibody (AT8)</td>
			<td>Thermo Fisher</td>
			<td>MN1020</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Anti-early endosome antigen 1 (EEA1) antibody</td>
			<td>abcam</td>
			<td>ab2900</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 680 goat anti-mouse IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A21058</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>Alexa Fluor 790 donkey anti-rabbit IgG (H+L) secondary antibody</td>
			<td>Thermo Fisher</td>
			<td>A11374</td>
			<td>antibodies</td>
		</tr>
		<tr>
			<td>iBlot2 Dry Blotting System</td>
			<td>Thermo Fisher</td>
			<td>IB21001</td>
			<td>Gel transfer</td>
		</tr>
		<tr>
			<td>iBlot2 Transfer Stacks, Nitrocellulose, mini</td>
			<td>Thermo Fisher</td>
			<td>IB23002</td>
			<td>Gel transfer</td>
		</tr>
	</tbody>
</table>

enrichment of detergent-insoluble protein aggregates from human postmortem brain

I denne undersøgelse beskriver vi en forkortet trinvis fraktionering protokol for berigelse af detergent-uopløselige protein aggregater fra postmortem menneskehjernen. Den ioniske vaskemiddel N-lauryl-sarcosine (sarkosyl) solubilizes effektivt indbygget foldede proteiner i hjernevæv tillader berigelse af detergent-uopløselige protein aggregater fra en bred vifte af neurodegenerative proteinopathies, såsom Alzheimers sygdom (AD), Parkinsons sygdom og Amyotrofisk lateral sklerose og prionsygdomme. Menneskelig kontrol og AD postmortem hjernen væv blev homogeniseret og aflejret af ultracentrifugering ved tilstedeværelse af sarkosyl at berige detergent-uopløselige protein aggregater herunder patologiske fosforyleres tau, den kernekomponent af neurofibrillary tangles i Annoncen. Western blotting demonstreret differential opløseligheden af aggregerede fosforyleret-tau og de vaskemiddel-opløseligt protein, tidlig Endosome Antigen 1 (EEA1) i kontrol og AD hjernen. Proteom analyse afslørede også berigelse af β-amyloid (Aβ), tau, snRNP70 (U1 - 70 K), og apolipoprotein E (APOE) sarkosyl-uopløselige brøkdele af annonce hjerne i forhold til de af kontrol, i overensstemmelse med tidligere væv fraktionering strategier . Således, denne enkle berigelse protokol er ideelle til en lang række eksperimentelle applikationer lige fra Western blotting og funktionelle protein Co sammenlægning assays til massespektrometri-baseret proteomics.

Forkortet trinvis sarkosyl-fraktionering protokol blev brugt til at berige detergent-uopløselige protein aggregater fra kontrol og AD postmortem hjernen (figur 1). Proteiner fra TH-S, S1, S2 og P2 fraktioner var løst af SDS-PAGE, fastsat til 15 min i Coomassie blå Fikseringsvæske buffer efterfulgt af blid farvning med Coomassie strålende blå G-250 farvning buffer. Resuspension trin er valgfrit, da der var målbart niveau af protein i S2 fraktion (Se 1.1...

Watch this Scientific Journal Video about Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain at JoVE.com

Enrichment of Detergent-insoluble Protein Aggregates from Human Postmortem Brain

En forkortet fraktionering protokol for berigelse af detergent-uopløselige protein aggregater fra postmortem menneskehjernen er beskrevet.

Berigelse af Detergent-uopløselige Protein aggregater fra Postmortem menneskehjerne

In this study, we describe an abbreviated single-step fractionation protocol for the enrichment of detergent-insoluble protein aggregates from human ...

Research

JoVE Journal

Biochemistry

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Protein Complex Affinity Capture fra Cryomilled pattedyrsceller

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

Tandem affinitetsoprensning af proteinkomplekser fra eukaryote celler

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Protein fordøjelse, ultrafiltrering og størrelse udstødelse kromatografi at optimere Isolation af Exosomes fra humant blod Plasma og Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Kalibrering-fri In Vitro kvantificering af Protein Homo-oligomerisering ved hjælp af kommercielle instrumentering og gratis, Open Source lysstyrke analyse Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

Påvisning af proteinubiquitinationssteder ved peptidberigelse og massespektrometri

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Robust sammenligning af Protein niveauer på tværs af væv og udvikling ved hjælp af standardiserede kvantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Enkelt trin berigelse af en TAP-Tagged Histon Deacetylase af Filamentous svamp Aspergillus nidulans for enzymatisk aktivitetsanalyse

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Kinasehæmmer screening i selv monterede humane protein mikrosystemer

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

Karakterisering af individuelle protein aggregater ved infrarød Nanospektroskopi og atomkraften mikroskopi

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Oprettelse af meget specifikke kemisk induceret protein Dimerization systemer ved trinvis Phage udvælgelse af en kombinatorisk enkelt domæne antistof bibliotek