Bio-Rad Bio-Gel® HTP Hydroxyapatite User Manual
Page 11
When the bed is stable, pass at least two bed volumes of
starting buffer through the column.
Section 5
Applying the Sample
A load between 1 and 5 mg of protein per ml bed vol-
ume is normally used, although much larger amounts
have sometimes proved satisfactory. Phosphate buffers
are used with a stepwise or gradient increase in concen-
tration while the pH is held constant. Generally speaking,
the higher the phosphate concentration the less strongly
the proteins are adsorbed.
Initial experiments are conveniently performed by
adsorbing the protein in 10 mM buffer and eluting the
sample with stepwise concentration increases of about
two-fold. If the effluent is not monitored, at least one and
one-half column volumes of buffer should be used for
each step to provide complete elution of each peak. The
void volume of hydroxyapatite is about 75% of the bed
volume.
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