Making an Affinity Column
Making an affinity column
The column couples free alkyl or amino groups. Our favorite buffer, Tris, will therefore couple to the column VERY effectively. If the slightest amount of Tris is in your protein the coupling will fail. If you use dialysis to remove Tris beware, as it will reduce but not remove the Tris. We have found that the only effective way to have no Tris is to not add it in the first place. If you are purifying your proteins on some sort of affinity matrix, such as Maltose, or glutathione, simply substitute a good buffer for the tris in your affinity protocol. For example, we usually use the NEB pMal vectors which generate maltose binding protein fusions. The cell lysis, wash, column loading washing and elution is all performed using buffers made with MOPS or HEPES rather than Tris. I'm sure just the wash and elution buffers alone being good buffers would also be fine. You then dialyse away the maltose and salt with 0.1M MOPS and you are ready to go. The pH of the final MOPS dialysis will depend on whether you use Affigel 10 or 15 (see BioRad instructions).
Detailed instructions on performing the coupling of your protein in 0.1 M MOPS to the matrix come with the product. We use about 0.5 ml (packed volume) of matrix, which can bind up to 20 mg of protein, and couple for 4 hours at 4 degC. You test the efficiency of coupling by comparing the free protein concentration before and after the coupling reaction. It normally approaches 80%.
See the instructions with the product, less stable peptides may need much shorter incubation times.