Bio-Rad Foresight™ Chromatography Columns, Prepacked - Manual (Page 29)

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3.6.3 Sample Loading
a) Volume and Concentration
• Load 300 μl of sample per well, cover the
plate with a sealing film, and incubate it in
the microplate shaker at 1100 rpm, for a
predetermined incubation time
• The incubation time depends on the
type of study and the target capacity
utilization. For isotherm studies, one hr
and up to six hr may be needed to reach
equilibrium. For uptake kinetic studies
1–2 hr are recommended as the longest
incubation time. For any other study an
incubation time of 30–60 min usually
suffices
• A volume of 300 μl translates to a phase
ratio per well of 15 for the 20 μl media
plates, respectively. These phase ratios
generally lead to a decrease in the liquid
phase concentration of less than 80%,
which minimizes the experimental error
b) Collecting Unbound Material
• Remove the depleted sample using
centrifugation. A cycle time of at least
2 min at 300 g usually removes all the
liquid. Do not use vacuum filtration for this
step. After collecting the liquid, label the
collection plate “Flowthrough”, blot the
media plate on a paper towel, and place
it on top of a new collection plate
• The Flowthrough collection plate can now
be analyzed, for instance for total protein
concentration