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

5. Allow the sample to completely run into the column. Discard the first 3.0 ml
eluted.
6. Add 4.0 ml of the application buffer to elute the serum, while collecting
the 4.0 ml fraction from the column.
7. Wash the Econo-Pac 10DG column with 20 ml of the application buffer if
the column is to be used again immediately. If the column is to be stored,
wash with 20 ml of water containing 0.02% sodium azide.
1.6 Standard Serum IgG purification
1. Discard the buffer above the top frit of an Econo-Pac serum IgG purifica-
tion column and snap off the bottom tip.
2. Prewash for first time use only: Wash the column with 40 ml of regenera-
tion buffer* (fill the column twice), follow by 40 ml of the appropriate
application buffer (human or rabbit). Allow the buffer to drain to the top of
the frit. The column will not run dry. Proceed with step 4.
*Caution: Contains sodium thiocycnate. Avoid skin contact. Avoid breath-
ing dust or fumes.
[If the column has already been prewashed proceed directly to step 3.]
3. Equilibrate the column with 30 ml of application buffer.
4. Apply prepared sample to the column. See label on column package for
exact column serum capacity.
5. Elute the IgG with 20 ml of application buffer. For a more precise collection
method, collect fractions of volumes approximately equivalent to that of the sam-
ple applied. Determine the absorbance at 280 ml of each fraction using a spec-
trophotometer and combine effluent tubes containing the unbound protein peak.
6. An optional step: Most of the bound albumin can be eluted by washing the
column with application buffer containing 1.4 M NaCl.
7. Regardless of whether or not step 6 was carried out, regenerate the column
with 20 ml of regeneration buffer.
8. Wash the column with 30 ml of application buffer and continue with step 4
for the next chromatography cycle, or store the column at 4 °C in applica-
tion buffer containing 0.02% sodium azide.
1.7 Answers to Common Questions
1. How do I purify polyclonal IgG from sheep or goat serum?
The Econo-Pac serum IgG purification kit can be used for goat and sheep
serum. In these cases, the rabbit buffer will be suitable.
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