When working with antibodies, raw sera or ascites can be an extremely useful reagent without further purification. However, many applications require a higher level of purity. Antibodies can be purified by affinity chromatography using immobilized bacterial proteins such as protein A or Protein G. However, as these proteins bind non-specifically to the Fc region of antibodies, all antibodies present in the sample will be purified along with the specific antibody of interest. In polyclonal samples of antibody, this can lead to the presence of a significant amount of non-specific antibodies in the resulting sample.
Probably the most effective technique for obtaining specific purified antibody is the use of immunoaffinity chromatography. This technique relies on immobilizing the specific immunogen to which an antibody was generated on a matrix such as agarose beads. This matrix is then packed into a chromatography column and the sera or ascites is run over the column. The specific antibodies present in the sample bind reversibly to the immobilized immunogen and other antibodies; proteins and contaminants are washed from the matrix. The antibody is then eluted from the column, usually using a low (or high) pH buffer and neutralized. This simple process results in a highly specific, purified antibody to the immobilized peptide or protein.
For most researchers, the most difficult part of immunoaffinity purification is the preparation of the purification column. Typically, most labs do not possess either the equipment or expertise to prepare the immunizing peptide in house, but rather have it synthesized commercially. As a result, most labs simply are not prepared to conjugate peptides or proteins to an immobilization matrix. The simple solution to this dilemma is a product like Thermo Fisher Pierce’s Sulfolink Coupling Resin.
Sulfolink consists of a porous, cross-linked, 6% beaded agarose. The agarose carries an activated iodoacetyl group capable of reacting with reduced sulfhydryl groups. This reactive group is separated from the agarose bead by a 12 atom spacer. The chemistry is such that when the reduced sulfhydryl interacts with the iodoacetyl group, hydrogen iodide (HI) is release and a thioester bond is formed between the 12-atom spacer and the peptide or protein.
This simple procedure makes production of immunoaffinity columns easy and straight forward. When generating synthetic peptides, it is a simple matter to add a terminal cysteine through which the peptide can be linked to the resin. In cases in which the cysteine is not fully reduced, which can be determined by an Ellman’s assay, the peptide can be treated with TCEP (Tris(2-carboxyethyl)phosphine). TCEP is a reducing agent which will not interfere with the subsequent reaction and thus does not need to be removed or accounted for when doing the actual conjugation.
For those researchers needing to purify antibodies or other molecules binding specifically to a protein or peptide sequence, the Sulfolink Resin offers an excellent option for quick and straightforward production of affinity chromatography columns. The resin is available either by itself or in the form of kits containing all the necessary ingredients for immobilizing either peptides or proteins, including TCEP reducing agent and disposable chromatography columns.