(101d) Surface Isoelectric Focusing (sIEF) for Qualitative Protein Separation

Surface Isoelectric Focusing (sIEF) for Qualitative Protein Separation

Zhichao Wang, Adrienne R. Minerick

¹Department of Chemical Engineering, Michigan Technological University, Houghton, MI, 49931, USA

A novel surface isoelectric focusing (sIEF) technique for micro-scale IEF applications is described and utilized to illustrate broad and narrow range ampholyte separations. sIEF is capable of resolving ampholytic molecules such as proteins and peptides from a complex mixture at micrometer scale, which is 100 times smaller than the smallest macrochannel-IEF previously reported. This smaller scale allows sIEF to be seamlessly integrated with protein array libraries. Moreover, the surface-printed property of gel will make it feasible for spot recovery or orthogonal MS, TOF and other secondary analysis.

With sIEF, 40 to 60 μm wide micro-gel line are printed spanning a 300 μm gap between micro-patterned parallel gold electrodes via surface patterning tools (SPTs, Bioforce Nano eNabler^TM) on traditional microscope glass slides. Pharmalyte^TM carrier ampholyte solution in either broad range (pH 3-10) or narrow range (pH 6.7-7.7) are co-printed with the acrylamide monomer solution and then polymerized in-situ. Mixtures of green fluorescent protein (pI ~6.0) and r-phycoerythrin (pI ~4.4) as well as mixtures of fluorescent peptides pI markers (contains pI 6.8, 7.2 and 7.6) were then printed over the gel surface. Upon electric field applications in the 3 to 9 V range, separation performance tests were quantified. To further optimize the resolving ability of sIEF, dielectric surface coatings were employed to isolate and suppress electrode electrochemical reactions. Also additive chemicals were coprinted with the sIEF gels to reduce protein-protein and/or protein-surface interactions that improve band distortion challenges.

Our results illustrate the development of a 100 pL gel for surface IEF-PAGE with easily adjustable pH gradient ranges. This new sIEF approach has lower applied voltages and smaller sample requirements, relatively quick fabrication process and device reusability, which make it more attractive as a portable, user-friendly platform for qualitative protein separations in a wide variety of protein analysis applications.