SDS-PAGE gel for protein-polymer complexation and release studies.

20 μL of different samples were mixed with 5 μL of loading buffer and 20 μL of each sample was loaded on acrylamide gel. The protein-polymer mixture was prepared by incubation of the polymer and boronic acid modified protein for 12 h.

For complexation kinetics, the polymer and the protein were mixed in a centrifuge tube for different times. At the desired time, the complex was mixed with loading buffer and was characterized with a run in the gel immediately.

For ROS responsive release experiment, identical protein-polymer conjugate samples (after incubation for 12 h) were treated with different amounts of H₂O₂ (1 mM or 10 mM) and incubated at room temperature for different time intervals, before subjecting to acrylamide gel electrophoresis. To calculate the amount of released protein from each sample, standard curves were generated from the known concentrations of pure protein samples loaded into the gel lanes. The gel image analysis and quantification were performed with Bio-Rad Image Lab TM software.

For redox and pH responsive release experiment, the protein-polymer complexes were treated with DTT (or GSH, 10 mM) and pH =5.0 for 12 h, before subjecting to acrylamide gel electrophoresis.