Difference between revisions of "Making an Affinity Column"
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Revision as of 23:44, 21 December 2009
We use affigel from Biorad as the column matrix, but many others are available and work well. It comes in two flavours, Affigel 10 (Biorad cat# 153-6046) and Affigel 15 (Biorad cat#153-6052). The different products prefer proteins with different pIs. You can either use your favorite software to tell you the pI of your protein and use the appropriate product, or be lazy and use a 1:1 mixture of both products. You can also buy a 1:1 mix (cat#153-6098).
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%.
Following coupling you should block any unreacted esters. This is easy. Remove your protein, or what is left of it, and replace with 1M ethanolamine pH8.0, mix at 4deg C for an hour. Rinse with TBS and store at 4degC in TBS plus 0.2% sodium azide.
There are many ways to couple peptides. The best choice depends on the sequence of your peptide, and which end you want linked to the matrix. If you want to link the amino end you can use a matrix similar to Affigel, but you usually use anhydrous coupling in DMSO. Details are supplied with the Affigel products. If you would like to link via a cysteine residue you can make a very stable thio-ether bond to Epoxy-Sepharose-6B (Pharmacia). This is a handy way to link the c-terminus to the resin, just add a cys to the end when it is made. Do the coupling to 1.0ml of preswollen Epoxy-Sepharose-6B in 0.1M NaHCO3 pH9.0 in a small reaction volume with constant mixing at 37deg C for 24 hours. Washing with 50 ml of 0.1M NaHCO3 pH9.0, then block unreacted groups on the matrix by incubating the 1.0ml of resin in 5.0ml of 0.1M 2-mercaptoethanol for a couple of hours with gentle agitation.
See the instructions with the product, less stable peptides may need much shorter incubation times.