Fig. 3: The physics of 3D diffusion dictate projection electrophoresis device design and inform image analyses.
From: 3D projection electrophoresis for single-cell immunoblotting
(a) Lane density (limited by minimum spacing between separation lanes Δwell) is dependent on separated protein xy bandwidth (σxy) to avoid microwell-microwell crosstalk. σxy in turn depends on 3D diffusion of the injected protein. Simulated data shown in the left schematic; measured OVA data shown in micrograph and intensity profile (representative of two independent separation gels). Scale bar represents 100 μm. (b) Throughput is a function of both lane density and usable gel area; separated protein bands parallel to the gel edges in a cross-sectional image of the gel show uniform migration across the gel. Scale bar represents 1 mm. (c) Theoretical maximum lane density at a spacing of 4σxy (~5% overlap between lanes) and 6σxy (~0.3% overlap between lanes). Maximum lane density is inversely proportional to σxy2. (d) Measured diffusional xy band broadening (Gaussian fit peak width σxy) from purified proteins initially partitioned into 32 μm microwells, as a function of in-gel diffusion time (tdiff). Left: σxy vs. tdiff. Right: σxy2 vs. tdiff with linear fits (σxy2 = σxy,02 + 2Dtdiff). After 10 s EP, we measure σxy < 30 μm for all proteins, suggesting that 200 μm microwell spacing is sufficient. Linear fitting yields OVA σxy2 = 32.5tdiff-198 (R2 = 0.75); BSA σxy2 = 14.3tdiff + 29.8 (R2 = 0.88); IgG σxy2 = 5.41tdiff + 153 (R2 = 0.42). (e) Modeled time for ovalbumin bands to diffuse into the neighboring lane, as a function of microwell spacing Δwell. (f) Modeled separation resolution for BSA and OVA, as a function of EP time and electric field strength. (g) Modeled Péclet number (defined as the ratio of the time to reach a BSA-OVA separation resolution of 1 to the time at which the OVA band is expected to diffuse into the neighboring separation lane). (h) Physics-driven postprocessing. For each confocal slice (BSA, 7%T gel), the original image, that after physics-driven postprocessing (deconvolution of a point spread function modeling 3D diffusion), and summed pre- and post-deconvolution intensity profiles for a 100 μm region surrounding each row of protein bands are shown. Each image pair is scaled to the maximum of the (higher-intensity) deconvolved image. Scale bar represents 50 μm. Source data are provided as a Source Data file.
