Bio-Rad Affi-Gel Protein A Media - Manual (Page 3)

Introduction
The Affi-Gel protein A MAPS II (Monoclonal Antibody
Purification System) kit provides a dramatic improvement in protein
A-agarose methods for purification of mouse IgG
1
from ascites
fluid. When Affi-Gel protein A agarose is combined with special-
ly optimized MAPS II binding, elution, and regeneration buffers, pro-
tein 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
Affi-Gel protein A agarose is purified protein A coupled to
crosslinked agarose beads, with an exclusion limit of greater than
10 M daltons, via chemically stable amide bonds. This coupling
chemistry, plus the stability of native protein A, results in excellent
resistance to denaturing agents such as urea, chaotropic salts such as
guanidine hydrochloride or potassium thiocyanate, and acid and base
(pH 2-11). Affi-Gel protein A agarose contains approximately 2 mg
of protein A/ml gel.
Principle
Protein A, from Staphylococcus aureus, has the property of
binding with high specificity to the Fc region of Ig from most mam-
malian species.
3 When coupled to agarose beads, protein A can be
used to purify IgG, to selectively remove IgG prior to analysis of
other Ig classes, or to absorb immune complexes to purify anti-
gens.
2
The affinity of IgG for protein A is not the same for all species.
For this reason MAPS II buffers have been developed to optimize
binding and recovery of mouse IgG
1
.
Comparison of IgG Recoveries
Protein A-agarose preparations have been used extensively to
purify IgG and IgG subclasses from a variety of mammalian species. 4
Currently, protain A-agarose is being widely used to purify mono-
clonal antibodies from mouse ascites fluid or culture medium super-
natents. However, the usefulness of protein A-agarose in this
1
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