Spectramax 34c microplate reader

Microplate wells were coated with unlabeled Protein A (Southern Biotech, Birmingham, AL, USA) at a concentration of 0.05 mg/mL in phosphate-buffered saline (PBS) at 37 °C for 1 h, and then blocked with 5% nonfat dry milk in PBS buffer. Crude plant extract and purified CMG2-Fc standards (at concentration ranging from 0–0.5 µg/ML, 50 µL each) were added to wells and incubated at 37 °C for 1 h. CMG2-Fc bound to Protein A on the plate was detected by adding 50 µL of horseradish peroxidase (HRP)-labeled goat anti-human IgG (Southern Biotech, 0.5 mg/mL) at concentration of 0.4 µg/mL and incubated for 1 h at 37 °C. Between each of these steps, microplates were washed with PBS with 0.05% v/v of Tween-20. Finally, the protein concentration was quantified by adding 100 µL of TMB substrate (Promega, Madison, WI, USA). Plates were incubated at room temperature for 10 min, followed by addition of 100 µL 1N HCl to stop the reaction. The TMB substrate reacts with HRP, allowing colorimetric detection of CMG2-Fc levels. The absorbance was measured at 450 nm with a Spectramax 34C microplate reader (Molecular Devices).

The total soluble protein concentration in crude was measured with Bradford essay using the Quick Start Bradford 1× protein dye reagent (Bio-Rad, Hercules, CA, USA) at 5× dilution. A standard curve was generated from bovine serum albumin (BSA) at concentrations from 0–0.5 mg/mL with increments of 0.05 mg/mL. The BSA standards, samples, and diluted samples were loaded into a 96-well microplate at a volume of 10 µL. The Bradford dye (190 µL) was then added to each well, allowing color to develop for 2 min prior to reading the absorbance measurement at 565 nm with a Spectramax 34C microplate reader (Molecular Devices, San Jose, CA, USA).