Bio-Rad Affi-Gel Protein A Media - Manual (Page 8)

pleted with only one column volume of buffer per step, making the
total procedure fast and easy.
2.2 Buffer Considerations
The affinity of IgG for protein A is not the same for all species.
Most mouse IgG
1
immunoglobulins have low affinity for protein A.
For this reason, the patented MAPS II buffer system has been devel-
oped to optimize binding and recovery of mouse IgG
1
. 1 When Affi-
Gel protein A is combined with the specially optimized MAPS II
buffer system, protein A capacity for mouse IgG
1
from ascites fluid
is 6-8 mg/ml gel. (This capacity is 8-10 times higher than that
obtained with published methods.
1,2 ) Using the MAPS II buffers, the
Econo-Pac protein A columns therefore have a mouse IgG
1
capac-
ity of 10-14 mg per column.
An alternative binding buffer is 10 mM sodium phosphate, 0.15
M sodium chloride, pH 8.2. A typical elution buffer is 0.1 M sodi-
um citrate, and a suitable regeneration buffer is 1.5 M sodium thio-
cyanate.
2.3 Sample Preparation
Bio-Rad’s Econo-Pac 10DG disposable desalting columns are
recommended for easy ascites or serum preparation with minimal
sample dilution. Each desalting column can prepare up to 3 milliliters
of raw ascites. The ascites sample is collected in 4 milliliters of
binding buffer, so sample dilution is minimized. An alternative is to
dilute ascites sample one to one with binding buffer, and then adjust
the pH to that of the binding buffer.
If your sample is tissue culture supernatant, it should be con-
centrated to approximately 5 mg immunoglobulin/ml and then dilut-
ed one to one with binding buffer.
2.4 Standard Mouse IgG 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.
3. Apply prepared sample to the column. See “Column
Performance” for recommended sample volumes.
4. Wash the column with 20 ml binding buffer.
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