Authors

M. Taylor; T. Banerjee; N. VanBennekom;K. Teter

Comments

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Abbreviated Journal Title

J. Vis. Exp.

Keywords

Immunology; Issue 59; Surface plasmon resonance; AB toxin; translocation; endoplasmic reticulum; cell culture; cholera toxin; pertussis toxin; Multidisciplinary Sciences

Abstract

AB toxins consist of an enzymatic A subunit and a cell-binding B subunit(1). These toxins are secreted into the extracellular milieu, but they act upon targets within the eukaryotic cytosol. Some AB toxins travel by vesicle carriers from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol(2-4). In the ER, the catalytic A chain dissociates from the rest of the toxin and moves through a protein-conducting channel to reach its cytosolic target(5). The translocated, cytosolic A chain is difficult to detect because toxin trafficking to the ER is an extremely inefficient process: most internalized toxin is routed to the lysosomes for degradation, so only a small fraction of surface-bound toxin reaches the Golgi apparatus and ER6-12. To monitor toxin translocation from the ER to the cytosol in cultured cells, we combined a subcellular fractionation protocol with the highly sensitive detection method of surface plasmon resonance (SPR) (13-15). The plasma membrane of toxin-treated cells is selectively permeabilized with digitonin, allowing collection of a cytosolic fraction which is subsequently perfused over an SPR sensor coated with an anti-toxin A chain antibody. The antibody-coated sensor can capture and detect pg/mL quantities of cytosolic toxin. With this protocol, it is possible to follow the kinetics of toxin entry into the cytosol and to characterize inhibitory effects on the translocation event. The concentration of cytosolic toxin can also be calculated from a standard curve generated with known quantities of A chain standards that have been perfused over the sensor. Our method represents a rapid, sensitive, and quantitative detection system that does not require radiolabeling or other modifications to the target toxin.

Journal Title

Jove-Journal of Visualized Experiments

Issue/Number

59

Publication Date

1-1-2012

Document Type

Article

Language

English

First Page

12

WOS Identifier

WOS:000209222600050

ISSN

1940-087X

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