Bio-Rad DEAE Affi-Gel Blue Gel - Manual (Page 5)

through desalting using the Bio-Gel P-6DG gel, or
through dialysis. The buffers listed in Table 1 have
provided complete plasminogen removal when tested,
but some variation in non-IgG protein binding and IgG
recovery may be encountered using different serum
preparations. For this reason, the unbound protein
fraction, ideally containing only IgG, should always be
tested for proteolytic activity as described above. If a
sample displays minor amounts of proteolytic activity in
the unbound protein fraction, it may be necessary to
modify the buffer. This experiment may also help to
determine conditions for even higher recoveries of IgG in
the unbound fraction.
Transfer a sample to 20 mM Tris-HCl, pH 8.0, and
apply it to a column equilibrated with the same buffer.
Elute the column with a sodium chloride gradient, and
collect fractions of the same volume as the sample
applied. The sodium chloride concentration at which
plasminogen elutes can be determined by testing the
effluent for proteases. Fractions free of protease can be
pooled, or a second serum sample can then be purified at
a sodium chloride concentration slightly lower than that
at which non-IgG protein is detected. For higher purity or
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10. Regardless of whether the albumin is eluted or not,
regenerate the column with 4 to 5 bed volumes of
regeneration buffer followed by 3 bed volumes of
application buffer.
11. Some loss of capacity due to small amounts of
protein which remain bound to the gel may be
evident after about five cycles. To compensate for
this, increase the gel-to-sample ratio by 20% for
subsequent cycles. For serum preparations, the
useful life of the gel is generally eight to ten cycles.
Note: The first one or two runs using the gel may show
low levels of eluted dye in the high salt peak. This dye
does not interfere with the usefulness of the gel and
should not appear in subsequent runs. Leaching of the
dye can be avoided by washing the column with the
prewash buffer.
Optimizing the Separation
Conditions
The end result is highly dependent on the pH and
ionic strength of the sample. It is crucial that serum is
transferred into the appropriate application buffer, either
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