Bio-Rad Affi-Gel 15 Gel - Manual (Page 9)

protein coupled per ml of gel (Figure 4). The efficiency of
coupling will vary with the protein and conditions of cou-
pling (Figure 1). Greater than 30 mg protein/ml gel may
be coupled, but efficiency will taper off. When maximum
total capacity is desired, a high concentration of ligand
should be chosen (50 to 60 mg protein per ml of gel).
When maximum efficiency is the goal, as would be the
case with ligand preparations of limited quantity, the lig-
and concentration should be in the range of up to 25 mg
protein per ml of gel. Unbound sample may be recovered
and reused without further treatment. Optimum coupling
efficiency is achieved when the total reaction volume is
between 1.5 and 4.5 ml per ml of gel bed.
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3.2 Temperature
Coupling at 4 °C is recommended whenever possible.
The slower reaction rate at this temperature will afford a
greater measure of control, and many ligands will have a
greater stability at 4 °C than at 20 °C.
3.3 Time
Coupling to the Affi-Gel 10 and 15 supports is rapid.
As shown in Figure 3, for the Affi-Gel support, about 75%
of the maximum binding achieved with gamma globulin at
pH 7.5 takes place within 30 minutes at 4 °C. Ninety per-
cent of the maximal coupling is achieved in an hour, and
within 4 hours the reaction is complete.
3.4 Ligand Concentration
The amount of protein coupled is proportional to the
amount of protein added to the gel up to about 30 mg
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