Bio-Rad Affi-Gel 15 Gel - Manual (Page 14)

• Affi-Gel support is more than 12 months old. Try new
material.
• The Affi-Gel support has been stored too warm.
• pH is not optimal. For Affi-Gel 10 gel, pH should be
near or below the pI of your ligand. For Affi-Gel 15
gel, it should be near or above the pI. Buffer concen-
tration should be at least 10 mM to maintain optimum
pH. If pI is not known, try test coupling at a range of
different pHs.
• A primary amino group, other than the ligand is pre-
sent; avoid Tris or glycine buffers.
• Ligand is not pure. For polyclonal IgG samples,
switch to Affi-Gel Hz support. For other samples,
increase purity of ligand by chromatography or
preparative electrophoretic methods.
• Aqueous coupling conditions provide less control
than anhydrous conditions. Switch to anhydrous con-
ditions if ligand solubility permits.
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Section 6
Monitoring for Protein Coupling
Soluble (unbound) protein remaining in the coupling
and wash buffers can be assayed by the Bio-Rad Protein
Assay (catalog number 500-0006) or by measuring
O.D.
280
. If measuring O.D.
280
is preferred, the pH of the
sample should be lowered with 10 mM HCl. N-hydroxy-
succinimide released during the coupling will absorb at
280 nm at neutral or basic pH. N-hydroxysuccinimide will
also interfere with the Lowry protein assay.
Section 7
Troubleshooting
Occasionally, the ligand will not bind to Affi-Gel 10
or 15 affinity support. If the ligand does not bind, or if you
get a low capacity column, there are a number of possible
reasons.
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