Bio-Rad Foresight™ Chromatography Columns, Prepacked - Manual (Page 32)

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Materials and Methods
1. Preparation of stock solutions:
Loading buffer: 0.02 M Na Acetate, pH 4.5 was
adjusted to a conductivity of 5.0 mS/cm with 0.02 M
Na Acetate, 5 M NaCl, pH 4.5
Stock protein solution: human IgG from Cohn fraction
II+III (Sigma catalog #G4386-25G) was solubilized in
the loading buffer to a final concentration of
3.0 mg/ml. The stock solution was thereafter sterile
filtered.
2. Preparation of test solutions:
Loading buffer was added proportionally into test
tubes containing the stock protein solution to prepare
10 test solutions with a hIgG concentration ranging
from 0.25 to 3.0 mg/ml. The tubes were vortexed prior
to use. The total hIgG concentration was determined
as follows: 200 µl of each of the test solutions was
loaded to a 96-well plate to determine its absorbance
at 280 nm. Next, the protein concentration was
calculated using the Lambert-Beer law with a fixed
mass extinction coefficient of 1.4 ml/mg/cm.
3. Generation of adsorption isotherm data using
Foresight chromatography media filter plate:
A 96-well plate containing Nuvia ™ cPrime ™ , a mixed-
mode resin was used. The sequence of steps was as
follows:
a) Apply 300 µl of loading buffer to each designated
well. Apply vacuum to drain the loading buffer.
Repeat twice.
b) Apply 300 µl of test solution to each designated
well. Secure the plate to microplate mixer and
agitate at 1100 rpm for one hr. Longer incubation
time may be required to reach equilibrium.
A conservative incubation time of 4–6 hr is
recommended for a broad range of buffer
conditions and target molecules.