Continuous protein concentration via free-flow moving reaction boundary electrophoresis

- A novel model FFMRB for continuous protein concentration.
- Successful prediction of processing capacity, fold and location of protein enrichment via FFMRB.
- Up to 125.7 mL/h maximum processing capacity, 95.5% recovery and 12.6 times enrichment factor.

In this work, we developed the model and theory of free-flow moving reaction boundary electrophoresis (FFMRB) for continuous protein concentration for the first time. The theoretical results indicated that (i) the moving reaction boundary (MRB) can be quantitatively designed in free-flow electrophoresis (FFE) system; (ii) charge-to-mass ratio (Z/M) analysis could provide guidance for protein concentration optimization; and (iii) the maximum processing capacity could be predicted. To demonstrate the model and theory, three model proteins of hemoglobin (Hb), cytochrome C (Cyt C) and C-phycocyanin (C-PC) were chosen for the experiments. The experimental results verified that (i) stable MRBs with different velocities could be established in FFE apparatus with weak acid/weak base neutralization reaction system; (ii) proteins of Hb, Cyt C and C-PC were well concentrated with FFMRB; and (iii) a maximum processing capacity and recovery ratio of Cyt C enrichment were 126 mL/h and 95.5% respectively, and a maximum enrichment factor was achieved 12.6 times for Hb. All of the experiments demonstrated the protein concentration model and theory. In contrast to other methods, the continuous processing ability enables FFMRB to efficiently enrich diluted protein or peptide in large volume solution.