392095 Surface-Enabled Isoelectric Focusing (sIEF) with Carrier Ampholyte Type pH Gradient
This works describes the first illustration of a newly developed, novel surface-enabled isoelectric focusing (sIEF) technique for micro-scale IEF applications. This new sIEF approach requires smaller applied voltages and eliminates the need for electrode reservoirs, which are required by traditional bulk gel IEF and macrochannel-IEF. Isoelectric focusing is capable of resolving proteins from complex mixtures based on small differences in their isoelectric point (pI). sIEF can be conducted at scales 100 times smaller than previously reported for macrochannel-IEF. Furthermore, the separation occurs in a gel printed on a surface, making it easily accessible for spot recovery and subsequent analysis.
To fabricate the sIEF gel, surface patterning tools (SPTs) available on a Bioforce Nano eNablerTM were utilized to print a 40 to 60 μm wide line of unpolymerized acrylamide solution spanning a 300 μm gap between micro-patterned, parallel gold electrodes on a traditional glass microscope slide. Broad range PharmalyteTM carrier ampholyte solution (pH 3-10) was co-printed with the acrylamide monomer solution and then polymerized in-situ. Surface printing parameters were optimized with respect to carrier ampholyte concentration (4% wt/v), polymerization time (3h), surface properties of glass slide (UV-ozone treatment for hydrophobicity) and printing chamber humidity (15%). The established pH gradient was examined by pH-sensitive fluorescent dye FITC, with pH insensitive dye TRITC as intensity profile normalizer. Green fluorescent protein (pI ~6.0) and r-phycoerythrin (pI ~4.4) were then printed over the gel surface using the Nano eNablerTMSPTs. DC voltages of 3V (100 V/cm) and 9V (300 V/cm) were applied across the 300 μm electrode gap to achieve protein IEF in the polymerized micro gel strip.
Our results describe the development of a 100 pL gel for surface-enabled IEF-PAGE. In addition, sIEF results illustrate optimization of the applied voltage, reduction of pH gradient drift, as well as both understanding and reducing the band broadening that occurs during focusing. In summary, this device operates in volumes 100 times smaller than previously reported macrochannel-IEF, and, requires less sample/reagent, relies upon a relatively quick fabrication process, and the surface is reusable. These attributes make it more attractive as a portable, user-friendly platform for widespread adoption and commercialization.
See more of this Group/Topical: 2014 Annual Meeting of the AES Electrophoresis Society