| Autor: |
Broce, Sean, Hensley, Lisa, Tomoharu Sato, Lehrer-Graiwer, Joshua, Essrich, Christian, Edwards, Katie, Pajda, Jacqueline, Davis, Christopher, Bhadresh, Rami, Hurt, Clarence, Freeman, Beverly, Vishwanath Lingappa, Kelleher, Colm, Karpuj, Marcela |
| Rok vydání: |
2016 |
| DOI: |
10.6084/m9.figshare.c.3611000_d4.v1 |
| Popis: |
RVFV NP characterization using an immunoprecipitation assay and proteinase K digestion (PK). Upper drawing: schematic illustration of the glycerol gradient profiles, which consist of two distinct sedimentation peaks representing putative assembly intermediates (peak I) and highly ordered assembled particles (peak II). (A) SDS-PAGE analysis of the radioactively labeled CFPS products. (B) Glycerol gradient fractions of the immunoprecipitated material resulting from a mixture of peaks I and II. (C) Glycerol gradient fractions of the immunoprecipitated material resulting from peak I. (D) Glycerol gradient fractions of the immunoprecipitated material resulting from peak II. The data strongly suggest that peak II represents native-like fully assembled capsids, whereas peak I represents assembly intermediates. (E) Radioactively labeled RVFV NP was translated for 1 h at 26 °C and equal amounts of RVFV NP from the middle fractions 8 and 9 (lanes 4 and 5) and the bottom fraction 22 (lane 6) from the glycerol gradients were loaded onto an SDS-PAGE gel and stained with Coomassie brilliant blue. (F) The same concentration of radioactively labeled RVFV NP from each fraction (lanes 1 and 2 represent fractions 8 and 9 from the glycerol gradients, and lane 3 represents fraction 22) was exposed to 100 μg/ml PK or (G) 250 μg/ml PK. As a negative control to the PK reaction, DDW was added to the same final volume as the PK. As a positive control for PK activity, 4 μg of BSA was added to 20 μl of each reaction. The PK digestion assay was performed for 2 h at 37 °C. The input of the RVFV NP was normalized before PK digestion. BSA was detected using Coomassie staining (Additional file 1: figure S1 e, f and g lower panel). (PDF 3164 kb) |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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