Thiophilic chromatography depends on the binding of proteins to sulphur atoms contained in a particular chemical context. During the development of methods for the preparation of affinity supports
it was noticed by Porath and colleagues (6), that agarose activated with divinylsulfone and capped with mercaptoethanol had a strong affinity for proteins (Fig. 1).
This affinity was greatest in the presence of a high concentration of lyotropic ions such as sulphate and was reversed at lower salt concentration (see Note 18). This provides a useful method for the purification of immunoglobulins as they have a stronger affinity than most other serum proteins. This method has found particular application for the purification of IgM and chicken immunoglobulin (IgY) and is described in more detail by Boschetti (7).
1. Equilibrate the thiophilic matrix (see Note 4) with five-column volumes of equilibration buffer (0.05 M Tris-HCl, 0.5 M potassium sulphate, pH 8.0). Commercial thiophilic matrices have a capacity of 15-20 mg protein per milliliter of gel.
2. Slowly add an equal volume of 0.1 M Tris-HCl, 1 M potassium sulphate, pH 8.0, to the gently stirring immunoglobulin sample (see Note 19).
3. To clarify the solution, centrifuge at 4000g, at 4°C for 20 min.
4. Apply the supernatant to the column and wash with 10-column volumes of equilibration buffer or until the absorbance of the eluate at 280 nm is less than 0.05.
5. Elute the immunoglobulin from the column with 0.05 M Tris-HCl, pH 8.0. Collect the eluate in suitable sized fractions (2 mL) and check the absorbance of these fractions at 280 nm. Pool the protein-containing fractions and dialyse against a suitable buffer (see Note 11).
Was this article helpful?