Bicinchonic acid bca protein assay kit

Particle size was measured using Nanotrac ULTRA instrument by suspending NPs in PBS while polydispersity index (PDI; i.e., the width of the particle size distribution) was calculated using the formula: (σ/d)² where σ represents the standard deviation and d indicates mean diameter. Zeta potential was measured using Zetasizer (Malvern Instruments Ltd, MA, USA). A known amount of NPs (0.25–0.5 mg) were resuspended in 1 ml distilled water and further diluted ten-times before measuring particle size and zeta potential. The quantity of Tag actually encapsulated was confirmed based on the amount of Tag (protein) extracted after degrading a fixed amount of NPs. 5 mg of NPs were degraded using 100 mM NaOH + 0.05% SDS (Sigma-Aldrich, MO, USA) by incubating at 37°C on a shaker. Samples were further centrifuged at 11,000 × g at 4°C for 10 min and the supernatants were tested for their protein content [22 (link)]. Protein estimations were done in triplicate using Bicinchonic acid (BCA) protein assay kit (Thermo Scientific, IL, USA) as per manufacturer’s instructions. Encapsulation efficiency was calculated as follows: amount of protein encapsulated / amount of protein used in encapsulation × 100%.