The encapsulated β-gal was released in its
active form; therefore,
β-gal activity in the supernatant was used to quantify the fractional
release of protein as a function of time, β-gal(t)/β-gal0 via colorimetry. β-Gal activity was
measured using ortho-nitrophenyl-β-galactopyranoside
(ONPG) according to the manufacturer’s protocol (Sigma-Aldrich).
Briefly, room-temperature ONPG (16 mM in 0.1 M phosphate buffer; pH
adjusted to 7.5) was mixed 2:1 with the β-gal test solution
in a 96-well plate (ONPG:β-gal test solution; 100 μL:50
μL). β-Gal catalyzes the hydrolysis of ONPG to release ortho-nitrophenol, a chromogenic substrate with maximal
absorbance at 420 nm. Absorbance at 420 nm was measured every 60 s
for 10 min, and the mean slope of the resultant curve was recorded.
To quantify β-gal(t)/β-gal0, the mean slope of the β-gal test solution at each time point
was compared to that of freshly prepared β-gal solution (200
μg/mL). β-gal(t) is the cumulative amount
of active β-gal released at time, t, and β-gal0 is the total amount of β-gal loaded into the gel initially.
active form; therefore,
β-gal activity in the supernatant was used to quantify the fractional
release of protein as a function of time, β-gal(t)/β-gal0 via colorimetry. β-Gal activity was
measured using ortho-nitrophenyl-β-galactopyranoside
(ONPG) according to the manufacturer’s protocol (Sigma-Aldrich).
Briefly, room-temperature ONPG (16 mM in 0.1 M phosphate buffer; pH
adjusted to 7.5) was mixed 2:1 with the β-gal test solution
in a 96-well plate (ONPG:β-gal test solution; 100 μL:50
μL). β-Gal catalyzes the hydrolysis of ONPG to release ortho-nitrophenol, a chromogenic substrate with maximal
absorbance at 420 nm. Absorbance at 420 nm was measured every 60 s
for 10 min, and the mean slope of the resultant curve was recorded.
To quantify β-gal(t)/β-gal0, the mean slope of the β-gal test solution at each time point
was compared to that of freshly prepared β-gal solution (200
μg/mL). β-gal(t) is the cumulative amount
of active β-gal released at time, t, and β-gal0 is the total amount of β-gal loaded into the gel initially.