Figure_3.tif (2.71 MB)

GFP-tagged SCP is incorporated specifically into virus particles.

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posted on 11.07.2012, 00:18 by Jens B. Bosse, Rudolf Bauerfeind, Leonhard Popilka, Lisa Marcinowski, Martina Taeglich, Christophe Jung, Hannah Striebinger, Jens von Einem, Ulrike Gaul, Paul Walther, Ulrich H. Koszinowski, Zsolt Ruzsics

(A) Gradient purified virus particles were immobilized on fibronectin-coated cover-slips, fixed and processed for immunofluorescence. A MCP specific polyclonal serum was used as indicator of virus capsids and a GFP specific polyclonal antiserum was used to compare MCP and GFP specific signals. Direct GFP fluorescence was detected by excitation with 488 nm laser light and appropriate emission filters. Inserts depict 2x magnifications. Scale bars represent 10 µm and 2.5 µm in inserts. (B) Quantification of particle fluorescence intensity distributions. Gradient purified S-GFP-SCP or S-mCherry-SCP virus preparations were bound on Poly-Lysin coated glass-bottom dishes and fluorescent spots were recorded in 16 bit mode. Exposure times as well as the EM gain were adjusted to maximize the recorded dynamic range. The graph depicts the integrated fluorescence distributions for S-GFP-SCP (black) and S-mCherry-SCP (red). (C) Immunoblot of gradient-purified virus particles. Approximately 1*105 PFU per lane of wt, (WT), S-GFP-SCP (S-GFP-SCP), S-mCherry-SCP (S-mCherry-SCP) or S-GFP-SCP* (S-GFP-SCP*) were spun down and lysed. Proteins were separated by SDS-PAGE, blotted and immunodetected with polyconal sera against SCP, GFP and mCherry. (D) Immunoelectron microscopy of purified nuclear capsids. Nuclear capsids were purified from wt (WT), S-GFP-SCP (S-GFP-SCP) or S-GFP-SCP* (S-GFP-SCP*) infected cells and immuno gold labeled with an antibody against GFP followed by protein A coupled to 10 nm gold. The scale bar indicates 50 nm. (E) Quantification of gold-labeling intensity. The amount of gold beads per capsid was counted for at least 20 views containing at least 20 capsids per condition as shown in (D). The mean as well as the standard deviation are depicted.


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