Microchamber Western Blotting Using Poly‑l‑Lysine Conjugated Polyacrylamide Gel for Blotting of Sodium Dodecyl Sulfate Coated Proteins

We report a novel strategy to immobilize sodium dodecyl sulfate (SDS)-coated proteins for fully integrated microfluidic Western blotting. Polyacrylamide gel copolymerized with a cationic polymer, poly-l-lysine, effectively immobilizes all sized proteins after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and enables SDS-PAGE and subsequent immuno-probing in an automated microfluidic chip. Design of a poly-l-lysine conjugated polyacrylamide gel allows optimization of SDS-protein immobilization strength in the blotting gel region of the microchamber. The dependence of protein capture behavior on both the concentration of copolymerized charges and poly-lysine length is studied and gives important insight into an electrostatic immobilization mechanism. Based on analysis of protein conformation, the immobilized proteins bind with partner antibody after SDS dilution. We demonstrate each step of the microchamber Western blot, including injection, separation, transfer, immobilization, blocking, and immunoblot. The approach advances microfluidic protein immunoblotting, which is directly relevant to the widely-used SDS-PAGE based slab-gel Western blot, while saving sample volume, labor, and assay time