Journal: Analytical Chemistry

Article Title: Microfluidic Western Blotting of Low-Molecular-Mass Proteins

doi: 10.1021/ac5024588

Figure Lengend Snippet: Figure 2. Optimization of discontinuous buffer system for low molecular mass PAGE. (A) PAGE kymograph of Tris glycine (top) and Tris tricine (bottom) discontinuous buffer systems in a 12% discontinuous gel. PAGE is operated under a fixed current of 1.5uA for Tris tricine and 1uA for Tris glycine, yielding ~25-55V/cm voltage ramp during each separation. (B) ITP sample stacking intensity profiles for protein ladder stack in open-channel regions for both the Tris glycine (upper) and Tris tricine (lower) systems at initial sample loading and minimum sample width. During stacking, a 1.5uA fixed current is applied for Tris tricine (~12-25V ramp) and a 0.3uA fixed current (~4- 8V ramp) for Tris glycine (as lower current yielded better stacking). Inset shows ITP stacking in a 4%T stacking gel for the Tris glycine system, added to reduce putative EOF-induced dispersion. (C) Inverted fluorescence micrographs and corresponding intensity profiles of sizing in the Tris glycine (top, open-channel loading no 4%T gel) and Tris tricine (bottom) systems. In both cases, the 25kDa ladder protein is at the 1.5mm separation distance position.

Article Snippet: To load sample onto the chip, 2.3μL of sample is pipetted into a well and electrophoresed into the channel at 1.5μA (~11V) (1.0μA, ~11V for Tris glycine) for 80 s. The well is then washed out with the terminating electrolyte run buffer consisting of 0.1% Triton X-100, 0.1% SDS, 3% DMSO with either 1X Tris glycine (25mM Tris, 192mM glycine Bio-Rad 161- 0734) or 1X Tris tricine (100mM Tris, 100mM tricine, SigmaAldrich T1165) and a fixed current applied across the channel to stack the injected plug, via transient isotachophoresis, and then size the sample species.

Techniques: Dispersion, Fluorescence

Journal: Analytical Chemistry

Article Title: Microfluidic Western Blotting of Low-Molecular-Mass Proteins

doi: 10.1021/ac5024588

Figure Lengend Snippet: Figure 3. A larger pore-size gradient at the open-channel/gel interface reduces unwanted size-exclusion effects during prob- ing. (A) Inverted fluorescence micrographs show antibody probing across a gel with smaller pore sizes at the interface19 (left) and for a gel with a gradient to larger pore sizes at the interface (right), both with 12%T gels utilizing DHEBA cross-linker and 600nM purified PSA sample. Gel interface is marked with black arrow; expected location of the PSA major isoform is indicated with an (*). (B) In- verted fluorescence kymographs of a 116-6.5kDa ladder separation in an 8%T (top) and 12%T gel (bottom) with a Tris tricine discon- tinuous buffer. Right panel shows the ladder when the 25kDa marker is 1.5mm into the gel. In the 8%T gel, the small 6.5kDa marker migrates faster than the stack and so rejoins the stack a short distance into the gel. 12%T enables destacking and separation of full 116-6.5kDa ladder. (C) Schematic depicting fabrication proto- col yielding a short larger-to-bulk pore-size gradient at the separa- tion gel interface.

Article Snippet: To load sample onto the chip, 2.3μL of sample is pipetted into a well and electrophoresed into the channel at 1.5μA (~11V) (1.0μA, ~11V for Tris glycine) for 80 s. The well is then washed out with the terminating electrolyte run buffer consisting of 0.1% Triton X-100, 0.1% SDS, 3% DMSO with either 1X Tris glycine (25mM Tris, 192mM glycine Bio-Rad 161- 0734) or 1X Tris tricine (100mM Tris, 100mM tricine, SigmaAldrich T1165) and a fixed current applied across the channel to stack the injected plug, via transient isotachophoresis, and then size the sample species.

Techniques: Pore Size, Fluorescence, Purification, Marker