Table 2 Probe delivery gel fabrication parameters for electrophoretic and diffusive probing.
From: 3D projection electrophoresis for single-cell immunoblotting
Gel type | Final agarose concentration after probe dilution | Antibody probes | Gelation time | Fabrication setup |
|---|---|---|---|---|
Primary antibody electrophoretic probe delivery agarose gel | 1.5% (w/v) (dissolved in 1× tris-glycine) UltraPure LMP Agarose, (Thermo-Fisher: 16520050) | For the studies reported in Fig. 5 and Supplementary Figures 4–6, dilutions from each stock were: (1) Rb α-actinin (1:15, 6.67% (v/v)) & (2) Gt α-GAPDH (1:10, 10% (v/v)). For the studies reported in Fig. 1 and Supplementary Figure 8, dilutions from each stock were: (1) Ms α-PTBP1 (1:10, 10% (v/v)) & (2) Gt α-GAPDH (1:10, 10% (v/v)) | 5 mins on ice pack | Glass plate and glass slide |
Secondary antibody electrophoretic probe delivery agarose gel | 1.5% (w/v) (dissolved in 1× tris-glycine) UltraPure LMP Agarose, (Thermo-Fisher: 16520050) | For the studies reported in Fig. 5 and Supplementary Figures 4–6, dilutions from each stock were: (1) Dk α-Rb AF555 (1:10, 10% (v/v)) & (2) Dk α-Gt AF488 (1:10, 10% (v/v)). For the studies reported in Fig. 1 and Supplementary Figure 8, dilutions from each stock were: (1) Dk α-Ms AF555 (1:20, 5% (v/v)) & (2) Dk α-Gt AF488 (1:20, 5% (v/v)) | 5 mins on ice pack | Glass plate and glass slide |
hFAB diffusive probe delivery agarose gel | 1.5% (w/v) - dissolved in 1× tris-buffered saline solution with Tween® (1× TBST, from Cell Signaling Technology 9997 S 10× stock) UltraPure LMP Agarose, (Thermo-Fisher: 16520050) | 1:10 (10% (v/v)) dilution from stock of either: (1) Bio-Rad hFAB Rhodamine α-GAPDH or (2) hFAB Rhodamine α-tubulin | 5 mins on ice pack | Glass plate and glass slide |
