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Immunologie et infection

표면 Plasmon 공명하여 호스트 Cytosol에 독소를 Translocation의 감지

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

Summary

이 보고서에서는, 우리는 표면 plasmon 공명이 호스트 cytosol에 독소 항목을 검색하는 데 사용됩니다 방법에 대해 설명합니다. 이것은 매우 민감한 방법은 cytosolic 독소의 양에 양적 데이터를 제공할 수 있으며,이 독소의 범위에 적용할 수 있습니다.

Abstract

AB의 독소 subunit 효소와 세포 결합의 B subunit 1로 구성되어 있습니다. 이러한 독소는 세포 환경으로 분비하지만, 그들은 진핵세포 cytosol 내의 대상에 따라 행동하고 있습니다. 세포 표면에서 cytosol 2-4 들어가기 전에 endoplasmic reticulum (ER)에 소포 항공사에 의해 일부 AB 독소 여행. 응급실에서 독소와 이동의 나머지 부분에서 단백질 실시 채널을 통해 촉매 체인 dissociates은 cytosolic 목표 5에 도달합니다. 응급실에 독소 거래가 매우 비효율적 프로세스이기 때문에 translocated, cytosolic 체인은 감지하기가 어렵습니다 : 대부분의 내면 독소는 잠돌군 Zz 장치 및 ER 6에 도달 표면 바운드 독소의 작은 파편 때문에 전용 저하에 대한 lysosomes로 라우팅됩니다 -12.

응급실에서 교양 세포의 cytosol에 독소의 translocation을 모니터하기 위해, 우리는 highl과 subcellular 분획화 프로토콜을 결합표면 plasmon 공명 (SPR) 13-15의 Y 민감한 검출 방법. 독소 – 치료 세포의 플라즈마 막은 선택 후 해독제 체인 항체와 코팅 SPR 센서를 통해 perfused하는 cytosolic 분수의 컬렉션을 수 있도록 digitonin와 permeabilized 있습니다. 항체 – 코팅 센서 cytosolic 독소의 PG / ML 수량을 캡처하여 검색할 수 있습니다. 이 프로토콜로, 그것은 cytosol에 독소 항목의 속도론를 수행하고 translocation 이벤트에 대한 억제 효과를 특성화 수 있습니다. cytosolic 독소의 농도는 또한 센서를 통해 perfused되었습니다 체인 표준의 알려진 수량과 생성된 표준 곡선으로부터 계산하실 수 있습니다. 우리의 방법은 radiolabeling 또는 대상 독소 다른 수정을 요구하지 않는 빠른, 민감한, 그리고 양적 탐지 시스템을 나타냅니다.

Protocol

1. digitonin의 준비 microcentrifuge 튜브에 100 % 에탄올 500 μL를 추가하고 80 ° C 10 분에 대해 설정된 열 블록에 넣습니다. digitonin의 1 % 주식 솔루션을 생산하는 온수 에탄올 250 μL에 dig​​itonin 2.5 MG를 해산. 0.04 %의 digitonin의 작업 솔루션을 생성하려면, HCN 버퍼 960 μL (50 MM의 Hepes 산도 7.5, 150 MM NaCl, 2 MM CaCl 2, 10 MM N – ethylmaleimide, 그리고 테아제 억제제에 digitonin 주식 솔루션 …

Discussion

기존의 방법론 비교

우리 SPR 기반 translocation 분석은 호스트 cytosol에 독소 전송을 감지하는 빠른, 민감한, 그리고 양적 방법을 나타냅니다. 기술 radiolabeling이나 독소에 다른 수정을 요구하지 않으며, 그것은 체인 항체를 사용할 수있는 해독제에 대한 독소를 적용할 수 있습니다. cytosol에 독소 통로를 모니터링하는 기존 방법은 별도의 cytosol과 막 분수 11, 23, 27, 28으로 파?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

이 작품은 K. Teter에 NIH R01 부여 AI073783에 의해 투자되었다. 우리는 원고의 중요한 읽기위한 subcellular 분별화 프로토콜과 헬렌 Burress의 개발에 도움 박사 쉐인 메시 감사합니다.

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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