Name: purification and aggregation of the amyloid precursor protein intracellular domain
Uploaded: 2012-08-28T12:00:00
Description: Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Forfatterne ønsker å takke dr. Hui Zheng (Baylor College of Medicine) for APP cDNA. Dette arbeidet ble finansiert av NIH tilskudd R21AG031948 (DB, JMB), F30AG030878 (ESS), R01DK073394 (AFO), John Sealy Memorial Endowment Fund for Biomedical Research (AFO) og Jean C. og William D. Willis Neuroscience Research Endowment (ESS). JMB er en lærd i translasjonsforskning Scholar Program og medlem av University of Texas Medical Branch Claude E. Pepper Eldre amerikanere Independence Center (støttet av NIH Grants UL1RR029876 og P30-AG-024 832, henholdsvis).

<html> 1. Uttrykk av rekombinant APP Intracellulær Domain (AICD) <ol><li> Transform E. coli stamme BL21 med menneskelig AICD (rester 649-695 av APP, neuronal isoform nummerering) klonet inn vektor pGEX-4T-1 (GE Healthcare). Denne vektoren vil uttrykke AICD som den C-terminale rest av et fusjonsprotein av glutation-S-transferase (GST). Denne vektoren koder også en trombin cleavage sekvens for å lette fjerning av GST moiety. Detaljer om kloning AICD inn pGEX-4T-1 finner du i vår forrige publisering fire...

<ol>
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I denne protokollen har vi skissert en prosedyre for å oppnå svært ren AICD for strukturelle, biofysiske og biokjemiske analyser. Denne fremgangsmåten krever ikke avansert kromatografi utstyr og er derfor tilgjengelig for de fleste laboratorier. Andre grupper har renset AICD 5-7,16, inkludert GST-AICD 17-19, for biokjemiske / strukturelle analyser. Ulemper til tidligere protokoller er dårlig løselighet av AICD 16, mindre enn ideelle renhet 17, og kravet til størrelsen e...

<table border="1"><tbody><tr><td> Navnet på reagensen </td><td> Selskapet </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> pGEX-4T-1 </td><td> GE Healthcare </td><td> 28-9545-49 </td><td></td></tr><tr><td> Trombin </td><td> GE Healthcare </td><td> 27-0846-01 </td><td></td></tr><tr><td> Ampicillin </td><td> Fisher Scientific </td><td> BP1760 </td><td></td></tr><tr><td> Bradford protein assay reagens </td><td> Bio-Rad </td><td> 500-0002 </td><td></td></tr><tr><td> Coomassie blått </td><td> Bio-Rad </td><td> 161-0786 </td><td></td></tr><tr><td> IPTG (isopropyl-beta-D thiogalactopyranoside) </td><td> Sigma-Aldrich </td><td> I6758 </td><td></td></tr><tr><td> Glutation-agarose </td><td> Sigma-Aldrich </td><td> G4510 </td><td></td></tr><tr&gt; <td> p-aminobenzamidine-agarose </td><td> Sigma-Aldrich </td><td> A7155 </td><td></td></tr><tr><td> Komplett proteasehemmere cocktail </td><td> Roche </td><td> 11836170001 </td><td></td></tr><tr><td> Slide-A-Lyzer dialyse kassetter </td><td> Thermo Scientific </td><td> 66380 </td><td></td></tr><tr><td> Kromatografikolonner </td><td> Evergreen Scientific </td><td> 208-3367-050 </td><td></td></tr><tr><td> Emulgator </td><td> Avestin, Inc </td><td> EmulsiFlex-C3 </td><td> Sterkt anbefalt </td></tr><tr><td> Eppendorf Thermomixer </td><td> Eppendorf </td><td> 022670107 </td><td></td></tr><tr><td> Glimmer disker </td><td> Ted Pella </td><td> 50-12 </td><td></td></tr><tr><td> AFM cantilevers </td><td> Bruker </td><td> MSNL-10 </td><td></td></tr><tr><td> WSxM programvare </td><td> NANOTEC </td><td> N / A </td><td> Free nedlasting </td></tr></tbody></table>

purification and aggregation of the amyloid precursor protein intracellular domain

Amyloid forløper protein (APP) er en type I transmembrane protein forbundet med patogenesen av Alzheimers sykdom (AD). APP er preget av en stor ekstracellulære domene og en kort cytosoliske domene kalt APP intracellulære domenet (AICD). Under modning gjennom den sekretoriske vei kan APP spaltes av proteaser betegnet α, β og γ-secretases 1. Sekvensiell proteolytisk spaltning av APP med β og γ-secretases fører til fremstilling av en liten proteolytisk peptid, betegnet Aβ, som er amyloidogenic og kjernen bestanddelen av senile plakk. Den AICD er også frigjort fra membranen etter secretase behandling, og gjennom samhandling med Fe65 og Tip60 kan translocate til kjernen til å delta i transkripsjon regulering av flere målgener 2,3. Protein-protein interaksjoner som involverer AICD kan påvirke menneskehandel, behandling og cellulære funksjoner av holo-APP og C-terminal fragmenter. Vi har nylig vist at AICD kan aggregere i vitro, og denne prosessen er hemmet av AD-innblandet molekylær anstand ubiquilin-1 4. Samsvar med disse funnene, har AICD utsatt hydrofobe domener, og er egentlig uordnede in vitro 5,6, men det får stabil sekundær struktur når bundet til Fe65 7. Vi har foreslått at ubiquilin-1 hindrer upassende Inter-og intramolekylær interaksjoner av AICD, hindrer aggregering in vitro og in intakte celler 4. Mens de fleste studier fokuserer på den rollen APP i patogenesen av AD, er rollen AICD i denne prosessen ikke klart. Ekspresjon av AICD har vist seg å indusere apoptose 8, å modulere signalveier 9, og for å regulere kalsium signalisering 10. Over-uttrykk for AICD og Fe65 i en transgen mus modell induserer Alzheimers som patologi 11, og nylig AICD er påvist i BHi lysates av vestlige blotting ved bruk egnede antigen henting teknikker 12. Å lette strukturelle, biokjemiske og biofysiske studier av AICD, har vi utviklet en prosedyre for å produsere rekombinant store mengder av meget rent AICD protein. Vi videre beskrive en fremgangsmåte for å indusere in vitro aggregering av termiske AICD og analyse ved atomic force mikroskopi. Fremgangsmåtene beskrevet er nyttige for biokjemisk, biofysiske og strukturell karakterisering av AICD og virkninger av molekylære anstand for AICD aggregering.

Watch this Scientific Journal Video about Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain at JoVE.com

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Fremgangsmåte for storskala rensing av APP intracellulære domenet (AICD) er beskrevet. Vi beskriver også metodikk for å indusere<em&gt; In vitro</em&gt; AICD aggregering og visualisering av atomic force mikroskopi. Fremgangsmåtene beskrevet er nyttige for biokjemiske / strukturell karakterisering av AICD og virkninger av molekylære anstand for aggregering sin.

Rensing og aggregeringen av amyloid forløperprotein intracellulære domenet

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

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