Effects of ampholyte dissociation constants on protein separation in on-chip isoelectric focusing.
Numerical simulations are presented for ampholyte-based isoelectric focusing in 2D microgeometries. In this study, model proteins are focused in the presence of 25 biprotic ampholytes under an applied electric field. Each protein is considered as a simple polypeptide having ten charge states, while the biprotic ampholytes are selected to generate a shallow pH range of 6 to 9. Straight and contraction-expansion microchannels are considered here, and a nominal electric field of 300 V/cm is maintained for separation of proteins. Six distinct values of deltapKs between 1 and 3.5 are investigated for ampholytes to form pH profiles in a 1 cm long microchannel. Simulation results show that relatively larger values of deltapK(deltapK > 3) are required to form stepless pH profiles in the system. The peak heights and differential resolutions of focused proteins are much higher for lower values of deltapK for which a stepped pH profile is evident. For each protein, the time it takes for the two edges of a peak to merge increases linearly with deltapK, while the focusing time goes up exponentially with increasing deltapK. Both merging and focusing times of protein are higher for contraction-expansion microchannel than those of straight microchannel. For a particular value of deltapK, the contracted "Zoom" region of contraction-expansion channel is able to form more tightly focused bands than the expanded region.
Shim
Jaesool
J
School of Mechanical and Materials Engineering, Washington State University, Pullman, WA 99164-2920, USA.
Dutta
Prashanta
P
Ivory
Cornelius F
CF
D000665:Ampholyte Mixtures / Q000737:chemistry; D002021:Buffers; D003198:Computer Simulation; D004560:Electricity; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation* / Q000379:methods*; D007526:Isoelectric Point; D008962:Models, Theoretical; D036103:Nanotechnology / Q000379:methods; D016011:Normal Distribution; D011506:Proteins / Q000737:chemistry*; D012984:Software; D013997:Time Factors
D000665:Ampholyte Mixtures; D002021:Buffers; D011506:Proteins