As two of important physico-chemical parameters,the acidic and alkaline residues of protein are of evident significance to evaluation of protein property and design of relevant separation and analysis.However,there is still no electrophoretic method used for directly detecting free acidic and alkaline residues of protein.Herein,we developed the concepts of moving reaction boundary (MRB) [1] and MRB titration (MRBT) [2,3],relevant MRBT theory,and method of microchip electrophoresis for counting free acidic and alkaline residues of protein.In the MRBT,the MRB was created with acid or alkali and target protein immobilized via highly-crossed degree polyacrylamide gel (PAG).It was theoretically revealed that the number of free acidic or alkaline residues of protein was nearly as a function of MRB displacement in the electrophoresis-based titration system.As a proof of concept,seven model proteins were chosen for counting acidic or alkaline residues of protein via the developed MRBT method.The results showed that the numbers of free acidic and alkaline residues of proteins counted were in good agreement with those obtained from the relevant amino sequences in the NCBI database,demonstrating the feasibility of the developed concept,theory and technique.The general methodology of MRBT developed has potential application for inexpensive,facilitative and informative protein structure analysis of free acidic or alkaline residues of protein.