Bio-Rad Bio-Scale™ Mini Affi-Prep® Protein A Cartridges - Manual (Page 4)

Purification Protocol
1. Pack a suitable chromatography column with the
desired volume of the Affi-Prep protein A support.
2. Equilibrate the column with 5-10 bed volumes of
Affi-Prep MAPS II binding buffer. 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.
4. Wash the column with 10-15 bed volumes of binding
buffer to remove all of the unbound contaminating
components.
5. Elute the immunoglobulin with 5 bed volumes of
Affi-Prep MAPS II elution buffer. Elute with and
additional 10 bed volumes of elution buffer to insure
total removal of immunoglobulin. Neutralize the
eluted sample immediately after elution with 1 M
Tris-HC1. (Prolonged exposure of the purified
immunoglobulin fraction to acid pH should be
avoided.)
6. Regenerate the Affi-Prep protein A column with
50% methanol after every use. The column can be
washed with 0.1 N NaOH every 5-10 runs for a more
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Sample Preparation
Proper adjustment of the pH and ionic streng h of the
sample is critical for optimal binding. For best results,
the sample pH should be adjusted to 9.0, and the ionic
strength of the sample should approach that of the MAPS
binding buffer. This can be achieved by sample dilution,
dialysis, or buffer exchange using the Econo-Pac
® 10DG
desalting columns or Bio-Gel ® P-6DG gel filtration gel.
• Ascites fluid should be diluted 1:2 with binding
buffer. Higher concentrations of binding buffer can
enchance the binding of low affinity antibodies.
• Tissue culture supernatant should be concentrated to
approximately 5 mg immunoglobulin per ml, and
then diluted 1:2 with binding buffer. For large
volume samples where further dilution is not desired,
we recommend adding the dry binding buffer salts
directly to the sample instead of diluting the sample
with prepared buffer.
• All samples should be filtered through a 0.45 or 0.8
µm filter before loading onto the column.
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LIT230B 9/2/98 10:28 AM Page 2