Xylohexaose (Megazyme
Ireland) was labeled with the fluorescent dye 2-aminobenzamide (2AB,
Sigma-Aldrich), as previously described.11 (link) Xylohexaose-2AB (0.5 mM) was then incubated with KfXYS1 and 30 mM UDP-Xyl in HEPES sodium salt pH 7.3 for 12 h, before
pelleting and washing as described previously. Alternatively, xylan
microparticles generated via a large-scale synthesis were labeled
with 2AB after formation as follows: 0.2 mg of XX-type microparticles
was suspended in 100 μL of 0.5 M sodium cyanoborohydride (NaCNBH4, Sigma-Aldrich) and 0.25 M 2AB (Sigma-Aldrich). Reaction
pH was adjusted to between 5 and 5.5 with 1 M acetic acid. The mixture
was then incubated at 45 °C for 12 h. Following incubation, the
reaction was pelleted by centrifugation at 5,000g for 5 min, the supernatant was removed, and the microparticles were
washed 10× with 1 mL of dH2O. Control samples were
also prepared following the same procedure but lacking NaCNBH4 in the reaction mixture.
Xylan microparticles (∼20
μg) were labeled with propidium iodide by incubation for 30
min in 100 μg/mL aq. propidium iodide (Sigma-Aldrich). Following
incubation, the microparticles were pelleted and washed with 50 μL
of water. The microparticles were then pelleted again, and the supernatant
was removed, before resuspension in 5 μL of 1% agarose solution
(60 °C), and mounting on a glass microscopic slide.
Ireland) was labeled with the fluorescent dye 2-aminobenzamide (2AB,
Sigma-Aldrich), as previously described.11 (link) Xylohexaose-2AB (0.5 mM) was then incubated with KfXYS1 and 30 mM UDP-Xyl in HEPES sodium salt pH 7.3 for 12 h, before
pelleting and washing as described previously. Alternatively, xylan
microparticles generated via a large-scale synthesis were labeled
with 2AB after formation as follows: 0.2 mg of XX-type microparticles
was suspended in 100 μL of 0.5 M sodium cyanoborohydride (NaCNBH4, Sigma-Aldrich) and 0.25 M 2AB (Sigma-Aldrich). Reaction
pH was adjusted to between 5 and 5.5 with 1 M acetic acid. The mixture
was then incubated at 45 °C for 12 h. Following incubation, the
reaction was pelleted by centrifugation at 5,000g for 5 min, the supernatant was removed, and the microparticles were
washed 10× with 1 mL of dH2O. Control samples were
also prepared following the same procedure but lacking NaCNBH4 in the reaction mixture.
Xylan microparticles (∼20
μg) were labeled with propidium iodide by incubation for 30
min in 100 μg/mL aq. propidium iodide (Sigma-Aldrich). Following
incubation, the microparticles were pelleted and washed with 50 μL
of water. The microparticles were then pelleted again, and the supernatant
was removed, before resuspension in 5 μL of 1% agarose solution
(60 °C), and mounting on a glass microscopic slide.