Electrophoretic injection and reaction of dye-bound enzymes to protein and bacteria within gel

Three-dimensional (3D) cell cultures within gels are used to examine physiological reactions between cells, including bacteria and macromolecules such as enzymes. Using non-denaturing electrophoresis, an anionic Coomassie Brilliant Blue (CBB) dye successfully bound to enzymes such as trypsin and lysozyme, and reacted with a protein and a bacterium within a gel. Both CBB-bound trypsin and lysozyme retained their enzymatic activities and migrated toward the anode in non-denaturing electrophoresis. CBB-bound trypsin successfully digested the iron-binding protein, transferrin, within the gel. Furthermore, the activity of esterase extracted from the bacteria, Bacillus subtilis was analyzed by the non-denaturing electrophoresis containing both the bacteria and the CBB-bound lysozyme after the bacteriolysis of the bacteria by the addition of CBB-bound lysozyme. This method can be applied to deliver enzymes to organisms including bacteria within 3D cell cultures.