CP4-EPSPS protein expression levels in samples
Leaf samples were collected at the vegetative growth stage (V2) (Fehr et al. 1971), flowering stage (R1), podding stage (R3) and mature stage (R7). All samples were quick-frozen with liquid nitrogen and stored at -70 °C for the estimation of EPSPS protein.
The protein was determined by ELISA using EPSPS detection kits (Envirologix, Portland, USA). Ten milligrams of each leaf sample were suspended in 1 mL of phosphate-buffered saline containing Tween 20 (PBST buffer), which was supplied as part of the kit, and all procedures were performed according to the manufacturer’s instructions. The absorption was measured on a microplate reader (Infinite M200, Tecan Group Ltd., Männedorf, Switzerland) at 450 nm.