Bio-Rad CM Affi-Gel Blue Gel - Manual (Page 4)

8. 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-sam-
ple ratio by 20% for subsequent cycles. The useful life of
the gel is generally eight to ten cycles.
Protease Free Globulin Fraction from
Serum with CM Affi-Gel Blue Gel
A partially purified globulin fraction is often desirable in the
preparation of immunological reagents. This is commonly
achieved by a two-step procedure involving fractionation on
DEAE anion exchange chromatography followed by ammonium
sulfate precipitation.
1 Recovery of active antibody by this method
is generally about 65%, and detectable protease activity remains.
In fact, ammonium sulfate may serve to activate serum proteases.
2
Better recoveries (80–90%) and complete removal of protease are
achieved by using CM Affi-Gel blue affinity gel instead of DEAE
cellulose, followed by an ammonium sulfate precipitation as an
additional purification and concentration step to obtain a globulin
fraction free of protease and serum complement proteins. This
procedure is particularly useful when processing large volumes of
serum or when high yields (>90%) are desired.
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2. To insure that the prewash removed excess dye in the gel,
wash the gel with 2 bed volumes of 1.4 M NaCl and then
with running buffer. If the high salt wash is colored, repeat
the first pre-wash.
3. Pack the gel into a suitable column.
4. Apply the serum sample.
5. Wash the column with 2 bed volumes of running buffer at a
flow rate of 15–30 cm/hr. The effluent from this step con-
tains the serum proteins minus plasminogen and albumin.
Approximately 90% of the globulin will elute in one col-
umn bed volume.
6. The albumin can be eluted with 2 bed volumes 1.4 M NaCl
in running buffer. This is optional, and can be eliminated.
7. Whether the albumin is eluted or not, regenerate the column
with 2 bed volumes of regeneration buffer followed by 2
bed volumes of running buffer.
Note: The first one or two cycles of the gel may show low levels
of eluted dye in the high salt peak. This does not affect the func-
tionality of the gel.
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