Coomassie blue r 250 dye

Following TA and LF complexation, the TA-LF complex solution was centrifuged at 1000 rpm (Sorvall ST 8 Centrifuge) to obtain stable TA-LF self-assemblies. Ten μL of these complex solutions, were transferred onto a nitrocellulose membrane (Bio Rad, Hercules, CA, USA) and the samples were allowed to diffuse protein into the membrane and dry at room temperature. Following a rinse with DI water, the membrane was incubated in 5 mL of 0.25% Coomassie Blue R-250 dye (#20278, Thermo Fisher Scientific) (50:10:40 v% methanol:glacial acetic acid:DI water). The nitrocellulose membrane in Coomassie Blue solution was heated in a 1200 W microwave for 30 s After cooling to room temperature, the dye solution was drained, and the membrane washed twice with DI water, then de-stained in 50:10:40 v% methanol:glacial acetic acid:DI water solution without dye until it displayed a clear background. After a final rinse with DI water, the blue spot protein-stained membrane was stored in DI water at room temperature and image captured using a camera. This assay was performed in triplicate. Similarly, these TA-LF complexes were used for generating SDS-PAGE gels according to our previous protocol [22 (link)].

All ex vivo assay samples were subjected to SDS-PAGE analysis [12% (w/v) polyacrylamide gel, Bio-Rad, CA, USA] and stained with Coomassie blue R-250 dye (Thermo Fisher, USA). For immunoblotting, the proteins on polyacrylamide gel were electrotransferred to a nitrocellulose membrane, which was subsequently blocked in blocking buffer (PBS + 0.05% Tween 20 + 5% skim milk) at 4°C overnight. Then the membrane was incubated with goat anti-chicken IgY-horseradish peroxidase (SeraCare, 5,000-fold diluted in blocking buffer) at room temperature for 1 h. After the washing procedure (PBS + 0.05% Tween 20), the membrane was developed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher, USA).