Protein Protocols Protein Protocols

Antigen-Ligand Columns 1001
4. When the A 280 reaches a low value (approx 0.02), disrupt the ligand–IgG interaction by
eluting with dissociating buffer. Monitor the A 280, and collect the protein peak into tubes
containing 1 M Tris-HCl, pH 8.8 (120 µL/1 mL fraction) to neutralize the acidic dissociating
buffer.
5. Wash the column with PBS until the eluate is at pH 7.4. Store the column in PBS containing
0.1% azide. Dialyze the IgG preparation against a suitable buffer (e.g., PBS) to remove
glycine/Tris.
4. Notes
1. The use of subclass-specific antibodies or MAb allows the immunoaffinity isolation of
individual subclasses of IgG.
2. Coupling at pH 6.5 is less efficient than at higher pH, but is less likely to compromise the
binding ability of immobilized ligands (especially antibodies).
3. Check the efficiency of coupling by measuring the A280 of the ligand before and after
coupling. Usually at least 95% of the ligand is bound to the matrix.
4. Elution of bound substances is usually achieved by using a reagent that disrupts noncovalent
bonds. These vary from “mild” procedures, such as the use of high salt or high or low
pH, to more drastic agents, such as 8 M urea, 1% SDS or 5 M guanidine hydrochloride.
Chaotropic agents, such as 3 M thiocyanate or pyrophosphate, may also be used. Usually
an eluting agent is selected that is efficient, but does not appreciably denature the purified
molecule; this is often a compromise between the two ideals. In view of this, highly avid
polyclonal antisera obtained from hyperimmune animals are often not the best reagents
for immunoaffinity purification, as it may be impossible to elute the IgG in a useful form.
The 0.1 M glycine-HCl buffer, pH 2.5, will elute most IgG, but may denature some MAb.
“Pre-elution” of the column with dissociating reagent just before affinity chromatography
ensures that the isolated immunoglobulin is minimally contaminated with ligand.
5. The column will only bind to its capacity and therefore some IgG may not be bound;
however, this can be saved and passed through the column again. The main problem is
with back pressure or even blockage, but this can be reduced by diluting the sample by
at least 50% with a suitable buffer (e.g., PBS). Incubation of the IgG containing sample
with the ligand matrix is not always necessary, but this will allow maximal binding to
occur. Alternatively, slowly recirculate the sample through the column, typically at