Fig.2. (A) Quantitation of EGFP mass per CHO cell with western blotting and fluorimetry. Immunofluorescence of a gel stained with primary antibody against GFP. Lane 1 (red band) was injected with the lysate of 5×104 pDP3 cells, while lanes 2-7 contained incremented amounts of recombinant EGFP; lane 8 (green bands), molecular size calibration. (B) Fluorescence intensity of the bands of the gel in A as a function of loaded protein: each point corresponds to the intensity of the lane above it. The line is a least squares regression line fitted to the points in the linear range of the blot. The protein mass in the pDP3 lysate band (lane 1) is estimated from the projection of the band intensity (leftmost circle) onto the regression line, yielding for this experiment 0.2 pmols, or 4.0 amols per cell. (C) Fluorimetric analysis of cell lysates. Lysates of 106 pDP3 or non-expressing control cells in 100 μl of non-denaturing buffer were scanned in the CLSM in the same manner as recombinant EGFP (see Materials and Methods). Filled symbols show fluorescence (mean±s.d.) of lysates: •, pDP3 cells (n=12 scans of 8 different lysates); , non-expressing cells (n=7 lysates). The unity slope regression line through the pDP3 lysate data corresponds to 57 nM recombinant EGFP, yielding 5.7 pmols total mass, or 5.7 amols/cell, in good correspondence with the value 5.4±1.1 amols/cell obtained from averaging estimates from regression analysis of the lysates of the 12 experiments.
Image published in: Peet JA et al. (2004)
Copyright © 2004. Image reproduced with permission of the Publisher, The Company of Biologists Ltd.
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