C18 nova sep pak column

An autodigestion procedure was used to investigate the effect of the expression of FAEA on the release of cell wall HCAs on cell death. Callus cultures (0.2–0.5 g fresh wt) were ground in 0.1 M Na acetate extraction buffer pH 5.0, in the presence and absence of excess β-1-4-endoxylanase (1000 U/sample) (GC140 Genencor Inc), without additional substrate, and incubated at 28 °C for 24 to 72 h with shaking at 120 rpm. Following centrifugation, soluble extracts were loaded onto a reverse phase C₁₈ µNova Sep-Pak column (Waters Inc), eluted with 100% MeOH and analysed by HPLC as described above.
Digestibility parameters were also determined from in vitro gas production measurements. Freeze dried powdered cell suspension cells (1.0 g samples) were fermented in triplicate in 165-ml capacity serum bottles in 90 ml of bicarbonate buffered medium and were inoculated with rumen micro-organisms in 10 ml of clarified rumen fluid, at 30 °C for 48 h according to the pressure transducer technique of Theodorou et al. (1994 (link)). This technique quantified the increase in head-space gas pressure (and thus the gas volume) in closed batch cultures inoculated with rumen micro-organisms. The cumulative gas production profiles were used to calculate the initial rate of gas evolution over the first 6 h, the maximum rate of gas evolution, the time to maximum rate and the total gas volume.

Ferulic acid esterase activity was determined in soluble extracts from 0.5 g of fresh or frozen cell cultures. Cells were ground in a precooled mortar and pestle with 0.1 M sodium acetate, pH 5.0 extraction buffer containing 0.5% Triton. Extracts were incubated with 24mM ethyl ferulate (ethyl 4-hydroxy-3-methoxycinnamate) at 28 °C for 24 h. Following incubation, and centrifugation, soluble extracts were loaded onto an activated reverse phase C18 µNova Sep-Pak column (Waters), and eluted with 4 ml 100% methanol (MeOH). FAE activity was calculated from the amount of ferulic acid released determined by HPLC. One unit of FAE activity equals 1 µg ferulic acid released from ethyl ferulate at 28 °C in 24 h.
Heat-shocked cells were assayed in the same way as above, but prior to assay the cells were routinely heat shocked at 38 ± 1 °C for 2 h in a shaken incubator and then allowed to recover for 24 h at 25 ± 1 °C on an orbital shaker. At the end of the 24-hour period cells were assayed for FAE activity by HPLC. FAE activity was also determined by measuring the release of monomeric and dimeric ferulic acids from self-digested cell culture samples.