An alternative easy method for antibody purification and analysis of protein-protein interaction-using GST fusion proteins immobilized onto glutathione-agarose

Abstract

Immobilization of small proteins designed to perform protein-protein assays can be a difficult task. Often, the modification of reactive residues necessary for the interaction between the immobilized protein and the matrix compromise the interaction between the protein and its target. In these cases, GST is a valuable tag providing a long arm that makes the bait protein to be accessible to the mobile flow phase of the chromatography. In the present report, we used a GST-fusion version of the 8 kDa protein SPINK3 as the bait to purify anti-SPINK3 antibodies from a rabbit crude serum. The protocol for immobilization of GST-SPINK3 to glutathione agarose beads was modified from previously reported protocols by using an alternative bifunctional crosslinker (DSP) in a very simple procedure and by using simple buffers under physiological conditions. We concluded that the immobilized protein remained bound to the column after elution with low pH, allowing the re-use of the column for alternative uses, such as screening for other protein-protein interactions using SPINK3 as bait.