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

5. Elute the column with three volumes of application
buffer.
6. Apply the serum sample and elute with three bed
volumes of starting buffer. Collect fractions which
are approximately the volume of the applied sample.
7. Beginning with the first tube of unbound protein
peak, combine effluent tubes to give a total volume
equivalent to eight times the volume of the initial
serum sample.
8. For the isolation of other serum proteins, the column
may be eluted with a gradient of increasing salt
concentration. A final NaCl concentration of 0.5 M
is usually enough to elute all serum proteins except
albumin.
9. Most of the bound albumin can be eluted by washing
the column with 2 to 3 bed volumes of 1.4 NaCl in
application buffer, or with regeneration buffer.
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2. Transfer the serum sample to the buffer. This should
be done using Bio-Gel P-6DG for buffer exchange,
or through dialysis.
3. Rinse the gel on a Buchner funnel with a least 5 bed
volumes of pre-wash buffer. DEAE Affi-Gel Blue
gel has an excess of dye. This wash will elute
residual dye which might be eluted in serum protein
fractions. Continue rinsing with at least 10 bed
volumes of application buffer (Table 1) to insure that
the ionic strength is lowered. Note: If the alcohol
wash is done in a column, you may notice shrinkage
of the gel.
4. Prepare a column of DEAE Affi-Gel Blue gel using
the gel-to-serum volume ratio given on the bottle
label. Calculate the total volume of gel needed using
the initial serum volume, prior to buffer exchange or
dialysis.
The capacity of the gel is lot dependent, and will
range between 0.2 and 1 ml of serum per ml of gel.
The capacity for human and rabbit serum for a
specific lot of gel is printed on the label of the gel
bottle. For species other than human or rabbit, use
the lower capacity given.
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