Bio-Rad Affi-Gel 15 Gel - Manual (Page 8)

Table 2. Coupling Efficiency of Acidic and
Neutral-to-Slightly-Basic Protein Under Various
Coupling Conditions
Affi-Gel 10 Affi-Gel 15
Coupling Coupling
Coupling Buffer Efficiency (%) Efficiency (%)
Bovine Serum Albumin, pI 4.9
0.1 M MOPS, pH 7.5 14 80
0.1 M MOPS, pH 7.5 + 80 mM CaCl 90 ——
0.1 M MOPS, pH 7.5 + 0.3 M NaCl 22 47
0.1 M MES, pH 4.8 90 38
Human Globulin, pI 7.0 (average)
0.1 M MOPS, pH 7.5 83 40
0.1 M MOPS, pH 7.5 + 0.3 M NaCl 69 70
0.1 M NaHCO
3
, pH 8.5 80 70
11
In addition to its effect on coupling, the slight charge
associated with each gel may sometimes be exploited in
the affinity separation itself, for example, it may be used
to enhance binding of weakly sorbed material, or elution
of strongly absorbed materials. In such cases, it may be
preferable to use the Affi-Gel 10 support to couple an
acidic protein, or the Affi-Gel 15 support to couple a basic
protein. Coupling efficiency can then be enhanced by
manipulating the coupling conditions in either of two
ways. Select the coupling pH so that the protein has a
charge opposite that of the gel, or add salt to the coupling
buffer to minimize charge interaction (80 mM CaCl
2
may
be useful for coupling acidic proteins to the Affi-Gel 10
support and 0.3 M NaCl may be useful when coupling
basic proteins to the Affi-Gel 15 support).
2 Examples of
these manipulations are shown in Table 2. The more basic
or more acidic the protein the larger the observed effects
will be.
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LIT156B 6/17/98 10:41 AM Page 10