Journal: Nature Communications

Article Title: 3D projection electrophoresis for single-cell immunoblotting

doi: 10.1038/s41467-020-19738-1

Figure Lengend Snippet: ( a ) Confocal imaging of PAGE of fluorescently labeled protein ladder at 20 s elapsed separation time. Each ladder sample is injected from a 32 μm diameter microwell ( xy plane) with PAGE along the z -axis of the gel block. Summing the background-subtracted fluorescence intensities at each pixel in the xy separation lane region of each slice image (a 300 pixel, 102 μm square region centered on each microwell) yields a z -intensity profile for each lane, with peak-to-peak displacement Δ z and peak width σ (10%T PA separation gel containing 10% Rhinohide®). ( b ) 3D renderings and z -intensity profiles are plotted for (i) 10 s electrophoresis and (ii) 15 s electrophoresis in 10%T PAGs containing 10% Rhinohide® for comparison with the data for 20 s electrophoresis shown in ( a ). ( c ) The electrophoretic mobility of the proteins depends log-linearly on protein molecular mass. Each plotted point represents an electrophoretic mobility calculated from linearly fitting migration distance vs. electrophoresis time data from 11 to 17 segmented separation lanes in n = 5 7%T PA separation gels containing 10% Rhinohide® (5 electrophoresis times). Linear fitting yields log(M r ) = 1.7 × 10 4 μ EP + 2.25 ( R 2 = 0.89). ( d ) Electromigration distance depends linearly on electrophoresis time, thus proteins migrate at constant velocity during PAGE. Migration distances are plotted from protein bands originating from 11 to 17 nearby microwells in two independent 7%T PA gels containing 10% Rhinohide®; 33 mA constant current; 52 V/cm initial, 2 gels for each migration time. Linear fitting yields OVA migration = 16.7 t -63.42; BSA migration = 12.64 t -39.3; IgG migration = 3.69 t -11.54. ( e ) Separation resolution, R s , for BSA and OVA peaks in 10%T PAGE gels containing 10% Rhinohide®. Each point depicts the mean and standard deviation of the R s calculation from the median migration distances and peak widths from n = 4 independent separation gels. Source data are provided as a Source Data file.

Article Snippet: Acrylamide precursor solutions for the various gels were prepared by diluting 30% stock acrylamide/bis-acrylamide precursor (Sigma-Aldrich A3699) and Rhinohide® (ThermoFisher R33400) solution (to increase mechanical robustness of substrate-free gels) in ultrapure water (Millipore®) and 10× tris-glycine (Bio-Rad 1610734) where appropriate.

Techniques: Imaging, Labeling, Injection, Blocking Assay, Fluorescence, Electrophoresis, Migration, Standard Deviation