JoVE Journal > Immunology and Infection
Summary
Abstract
Protocol
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Immunology and Infection

Påvisning af toksin translokation i værtslandet cytosolen af ​​overflade plasmon resonans

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

Summary

I denne rapport beskriver vi, hvordan overfladen plasmon resonans bruges til at påvise toksinet indrejse i værten cytosol. Denne meget følsom metode kan give kvantitative data om mængden af ​​cytosole toksin, og det kan anvendes til en række af toksiner.

Abstract

AB toksiner består af en enzymatisk A subunit og en celle-bindende B subunit 1. Disse giftstoffer udskilles i det ekstracellulære miljø, men de handler på mål inden for eukaryote cytosolen. Nogle AB toksiner rejse med Blære luftfartsselskaber fra cellens overflade til det endoplasmatiske reticulum (ER), inden de kommer ind i cytosolen 2-4. I ER, at den katalytiske En kæde dissocieres fra resten af toksinet og bevæger sig igennem en protein-ledende kanal nå sit cytosolisk mål 5. De omplantes, cytosole En kæde er svær at opdage, fordi toksin menneskehandel til skadestuen er en yderst ineffektiv proces: De fleste internaliserede toksin er dirigeres til lysosomerne for nedbrydning, så kun en lille brøkdel af overflade-bundet toksin når Golgi apparatet og ER 6 -12.

At overvåge toksin translokation fra ER til cytosolen i dyrkede celler, vi kombinerede en subcellulære fraktioner protokol med highly følsom påvisningsmetode af overflade plasmon resonans (SPR) 13-15. Plasma membran af toksin-behandlede celler er selektivt permeabilized med digitonin, så samling af et cytosol fraktion, som efterfølgende perfunderet over en SPR sensor belagt med et anti-toksin A kæde antistof. Den antistof-belagt sensor kan fange og afsløre pg / mL mængder af cytosolisk toksin. Med denne protokol, er det muligt at følge kinetikken af ​​toksin indtræden i cytosolen og at beskrive hæmmende på translokation begivenhed. Koncentrationen af ​​cytosole toksin kan også beregnes ud fra en standardkurve genereret med kendte mængder af en kæde standarder, der er blevet perfunderet over sensoren. Vores metode er en hurtig, følsom, og kvantitative detektion system, der ikke kræver radioaktiv mærkning eller andre ændringer til målet toksin.

Protocol

1. Udarbejdelse af digitonin Tilsæt 500 μL af 100% ethanol til et mikrocentrifugerør og læg den i en varmeblok indstillet til 80 ° C i 10 min. Opløs 2,5 mg digitonin i 250 μL af den opvarmede ethanol til at producere en 1% stamopløsning af digitonin. For at generere en arbejdsgruppe løsning på 0,04% digitonin, der tilsættes 40 μL af digitonin stamopløsning til 960 μL af HCN buffer (50 mM HEPES pH 7,5, 150 mM NaCl, 2 mM CaCl 2, 10 mM N-ethylmaleimide, og en protease…

Discussion

Sammenligning med eksisterende metode

Vores SPR-baserede translokation analyse repræsenterer en hurtig, følsom, og kvantitativ metode til at påvise toksinet levering i værten cytosol. Teknikken kræver ikke radioaktiv mærkning eller andre ændringer af toksin, og det kan anvendes på alle giftstof, som en anti-toksin A kæde antistof er til rådighed. Eksisterende metoder til at overvåge toksin passage ind i cytosol er også afhængige af en subcellulære fraktioner protokol til partitio…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Dette arbejde blev finansieret af NIH give R01 AI073783 til K. Teter. Vi takker Dr. Shane Massey for bistand til udvikling af subcellulære fraktionering protokollen og Helen Burress til kritisk læsning af 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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