Bio-Rad Affi-Gel Protein A Media - Manual (Page 5)

1.5 Sample Preparation
Sample preparation is simplified by using the Econo-Pac 10DG
columns. Each Econo-Pac 10DG desalting column can process up to
3 milliliters of ascites fluid and the columns can be re-used. Two
Econo-Pac 10DG columns should be reserved for buffer exchange
at the end of the protein A purification protocol. It is also recom-
mended that one Econo-Pac 10DG desalting column be used for
each (different) ascites to avoid cross contamination.
To prepare up to 3 ml of ascites for purification on the Affi-Gel
protein a column:
1. Discard the buffer above the top frit of one Econo-Pac 10DG
column.
2. Add 20 ml of binding buffer to the column (fill to the top), and
snap off the bottom tip to start the column flowing.
3. Allow the buffer to drain to the top frit. The column will not run
dry. Flow will stop when the buffer level reaches the top frit.
4. Add 3.0 ml of ascites fluid to the column. If the sample is less
than 3.0 ml, add buffer to reach a total sample volume of 3.0
ml.
5. Allow the sample to completely run into the column. Discard
the first 3.0 ml eluted.
6. Add 4.0 ml of binding buffer to elute the ascites, while collect-
ing the 4.0 ml fraction from the column.
7. Wash the Econo-Pac 10DG column with 20 ml of binding buffer
if the column is going to be used again right away, or wash the
column with 20 ml of water containing 0.02% sodium azide for
storage.
1.6 Standard Mouse IgG
1
Purification
Procedure
1. Discard the buffer above the top frit of the Econo-Pac protein A
column.
2. Equilibrate the column with 10 ml binding buffer. Allow the
buffer to drain to the top frit. The column will not run dry. After
equilibration, the pH of the column effluent should be equal to
the pH of the binding buffer (pH 9.0).
3. Apply the prepared sample to the column. Maximum recom-
mended sample volume is 2.0 ml ascites fluid.
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