JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
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Immunologia e infezioni

Påvisning av giftstoffer translokasjon i Host cytosol ved Surface Plasmon Resonance

Published: January 03, 2012
doi:
10.3791/3686
, , ,
1Department of Molecular Biology and Microbiology,University of Central Florida

Summary

I denne rapporten beskriver vi hvordan overflaten plasmon resonans blir brukt til å påvise toksin innreise til verten cytosol. Denne svært følsom metode kan gi kvantitative data om mengden av cytosoliske toksin, og den kan brukes til en rekke giftstoffer.

Abstract

AB giftstoffer består av en enzymatisk A subenhet og en celle-bindende B-subenhet 1. Disse giftstoffer skilles ut i ekstracellulære miljøet, men de handle på mål i eukaryote cytosol. Noen AB giftstoffer reise med vesicle bærere fra cellen overflate til det endoplasmatiske retikulum (ER) før vi går i cytosol 2-4. I ER, til katalytiske En kjede spaltes fra resten av toksinet og beveger seg gjennom et protein-ledende kanal nå sitt cytosoliske mål 5. Den translocated, cytosoliske En kjede er vanskelig å oppdage fordi toksinet trafficking til ER er en svært ineffektiv prosess: De fleste internalisert toksinet er rutet til lysosomer for degradering, så bare en liten brøkdel av overflate-bundet toksinet når Golgi-apparatet og ER 6 -12.

For å overvåke toksin translokasjon fra ER til cytosol i dyrkede celler, kombinerte vi en subcellular fraksjonering protokoll med highly sensitive deteksjonsmetode av overflaten plasmon resonans (SPR) 13-15. Plasma membran av toksin-behandlede cellene er selektivt permeabilized med digitonin, slik samling av en cytosoliske brøkdel som deretter perfused over en SPR sensor belagt med en anti-toksin En kjede antistoff. Antistoff-belagte sensor kan fange opp og oppdage pg / ml mengder cytosoliske toksin. Med denne protokollen, er det mulig å følge kinetikken av toksin oppføring i cytosol og å karakterisere hemmende effekter på translokasjon hendelsen. Konsentrasjonen av cytosoliske toksinet kan også beregnes fra en standard kurve generert med kjente mengder av en kjede standarder som har blitt perfused over sensoren. Vår metode representerer en rask, følsom, og kvantitative deteksjon system som ikke krever radiolabeling eller andre modifikasjoner til målet toxin.

Protocol

1. Utarbeidelse av digitonin Legg til 500 mL av 100% etanol til en mikrosentrifuge tube og legg den i en varme blokk innstilt på 80 ° C i 10 min. Løs 2,5 mg digitonin i 250 mL av det oppvarmede etanol å produsere en 1% stamløsning av digitonin. For å generere en fungerende løsning på 0,04% digitonin, tilsett 40 mL av digitonin stamløsning til 960 mL av HCN buffer (50 mM Hepes 7,5 pH, 150 mM NaCl, 2 mM CaCl 2, 10 mM N-ethylmaleimide, og en proteasehemmer cocktail). …

Discussion

Sammenligning med eksisterende metodikk

Våre SPR-baserte translokasjon analysen representerer en rask, følsom, og kvantitative metode for å detektere toxin levering i verten cytosol. Teknikken krever ikke radiolabeling eller andre endringer av giftstoffer, og det kan brukes på alle toksin som en anti-toksin A kjede antistoff er tilgjengelig. Eksisterende metoder for å overvåke toxin passasje inn i cytosol også stole på en subcellular fraksjonering protokoll for å partisjonere celle ek…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Dette arbeidet ble finansiert av NIH tilskudd R01 AI073783 til K. Teter. Vi takker Dr. Shane Massey for bistand i utviklingen av subcellular fraksjonering protokollen og Helen Burress for kritisk lesing av manuskriptet.

Materials

Name of the reagent Company Catalogue number
Digitonin Sigma D141
Ethanol Acros 61509-0010
DMEM Invitrogen 11995065
Fetal Bovine Serum Atlanta Biologicals S11550
Ganglioside GM1 Sigma G7641
CTA Sigma C2398
PTS1 List 182
NHS (N-Hydroxysuccinimide) Pierce 24500
EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) Thermo Scientific 22981
Ethanolamine Sigma E0135
PBST Medicago 09-8903-100
Anti-CTA antibody Santa Cruz Biotech sc-80747
Anti-CTB antibody Calbiochem 227040
Anti-PTS1 antibody Santa Cruz Biotech sc-57639
Refractometer Reichert SR7000, SR7000DC
SPR sensor slides Reichert 13206060
Syringe pump Cole Palmer 780200C
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Tags

Toxin TranslocationSurface Plasmon ResonanceAB ToxinsCell-binding B SubunitVesicle CarriersEndoplasmic Reticulum (ER)Catalytic A ChainProtein-conducting ChannelCytosolic TargetToxin TraffickingLysosomesGolgi ApparatusSubcellular Fractionation ProtocolDigitonin PermeabilizationAnti-toxin A Chain AntibodyDetection Method

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Taylor, M., Banerjee, T., VanBennekom, N., Teter, K. Detection of Toxin Translocation into the Host Cytosol by Surface Plasmon Resonance. J. Vis. Exp. (59), e3686, doi:10.3791/3686 (2012).
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