Folate binding assay and folate quantification
Hela cells were passaged and cultured in RPMI 1640 folate-free media (Sigma, R1145), supplemented with 10% FBS, 3.7g/L sodium bicarbonate, 1X glutamax (Gibco, 35050-061) and 1X Antibiotic-Antimycotic. Cells were transfected with wildtype and variant GFP-CIC plasmids when reaching 50% confluency. 36-48 hours later, culture medium was replaced with PBS supplemented with 2% FBS and 50nM folic acid (Sigma, F8758). Cells were cultured for 1 hour in the presence of FA, after which they were dissociated with 0.25% trypsin. Dissociated cells were resuspended in TBST with 1% ascorbic acid (pH 3.0-3.5) and incubated at 95℃ for 10 minutes. Supernatant was collected after being centrifuged for 10 minutes at 14000rpm. NaOH (1M) was added to collected supernatant at a ratio of 1:15 to reach a neutral pH value.
Bovine folate binding protein (50ng/ul, Sigma, F0504-1MG) was printed on a 96 well plate with 1ul in each well, and the plate was then sealed and incubated overnight at 4℃. On the second day, HRP-FA was 2-fold serially diluted to create a standard curve. Samples and HRP-FA were mixed at a ratio of 4:1. The printed 96 well plate was washed with TBST, and the mixed samples and standards were added into the wells and incubated for 1 hour prevented from light. The plate was washed with TBST, and 50ul of ELISA substrate were added to each well. Signal intensities were imaged using Q-view equipment and quantified using the Q-view software.