Bio-Rad Affi-Gel Protein A Media - Manual (Page 6)

4. Wash the column with 20 ml binding buffer.
5. Elute the IgG with 10 ml of elution buffer. Elute the column
with an additional 20 ml of buffer to insure total removal IgG.
In most cases, the majority of the IgG will elute in the first 10 ml.
6. Wash the column with 10 ml of regeneration buffer, followed
by 10 ml of PBS containing 0.02% sodium azide for storage.
7. Set up an unused Econo-Pac 10DG column previously equili-
brated with 20 ml of PBS or other buffer suitable for storage of
immunoglobulin.
8. Apply 3 ml of the IgG-containing fraction collected in step 5 to
the prepared Econo-Pac 10DG column. Discard the first 3.0 ml
eluted.
9. Add 4.0 ml of appropriate buffer (see step 7) to the Econo-Pac
10DG column and collect the 4.0 ml fraction from the column.
This fraction contains the protein A purified IgG. For a more
precise collection method, elute the antibody with 8.0 ml of a suit-
able buffer and collect 1.0 ml fractions. Analyze the fractions to
determine which fractions to pool.
10. Repeat until the entire IgG-containing fraction has been buffer
exchanged.
11. The Econo-Pac 10DG column should then be washed with 20 ml
of deionized water. Store at room temperature with 0.02% sodi-
um azide for re-use.
1.7 Answers to Common Questions
1. Sensitivity of antibodies to low pH:
There are a few antibodies which are inactivated by low pH.
Inactivation can be avoided by collecting the eluted fractions
into a concentrated neutral buffer or buffer exchanging the
fraction using an Econo-Pac 10DG column. In cases of
extreme sensitivity, many immunoglobulins can be eluted at
pH 4-6 by adjusting the pH of the elution buffer with 10 N
NaOH.
2. Expected flow rate:
0.5-1.5 ml/min.
3. Column regeneration:
Regenerating the column after every use will increase the life-
time of the gel and help eliminate cross-over contamination of
IgG from one run to the next.
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