Author: Spence, Jennifer S.; Krause, Tyler B.; Mittler, Eva; Jangra, Rohit K.; Chandran, Kartik
Title: Direct Visualization of Ebola Virus Fusion Triggering in the Endocytic Pathway Document date: 2016_2_9
ID: tnaizwxo_30
Snippet: Cleavage of EBOV-GP was performed by incubating the virus with 250 g/ml thermolysin (Sigma) for 1 h for partially cleaved GP and 500 g/ml thermolysin for 2 h for fully cleaved GP at 37°C after the first ultracentrifugation step. Protease activity was quenched by addition of 1 mM phosphoramidon (Peptides International) and incubation on ice for 20 min prior to the second ultracentrifugation step through a sucrose cushion. The extent of cleavage w.....
Document: Cleavage of EBOV-GP was performed by incubating the virus with 250 g/ml thermolysin (Sigma) for 1 h for partially cleaved GP and 500 g/ml thermolysin for 2 h for fully cleaved GP at 37°C after the first ultracentrifugation step. Protease activity was quenched by addition of 1 mM phosphoramidon (Peptides International) and incubation on ice for 20 min prior to the second ultracentrifugation step through a sucrose cushion. The extent of cleavage was confirmed by immunoblotting with an anti-GP1 antibody. Viral membranes were labeled with self-quenching concentrations of DiD (Life Technologies). Virus (50 g) was incubated with 250 M DiD for 90 min with gentle agitation in the dark at room temperature. Labeled virus was passed through a NAP-5 column (GE Healthcare) to remove excess dye and large aggregates and was used for imaging within 48 h. DiD labeling reduced viral infectivity by less than 10% (data not shown).
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