Three antibody possibilities: After immunization with the phospho peptide, three types of antibodies can be produced in the rabbit’s immune response. As seen in Figure 1 below, the first is the phospho-specific antibody that is desired. However, antibodies that are specific for the dephospho form of the peptide can also be generated if phosphatases in the rabbit dephosphorylate the peptide conjugate that had been injected. Lastly, pan-specific antibodies can be generated against sequences/conformations of the peptide that do not involve the phosphoryl group in the epitope. These antibodies react with the protein irrespective of its phosphorylation state. The only way to isolate the desired phospho-specific antibody is through sequential phospho- and dephosphoaffinity chromatography.
Isolation of the Phospho-Specific Antibody: The first column we use is the phosphopeptide affinity column (see Figure 2). We first prepare an IgG fraction from the serum and apply it to our phosphopeptide affinity column. Two of the three types of antibodies that we described previously will bind to this column. First, the phospho-specific antibody (blue) will bind because the phosphopeptide is present on this column. The pan-specific antibody (red) will also bind because the sequence of the peptide that it recognizes is also present on this column. The one antibody that will not bind to this column is the antibody that is specific for the dephospho form of the peptide (green), as this form of the peptide is not present on the column. These latter antibodies pass through the column in the flow-through, together with the entire complement of additional IgGs that were isolated from the rabbit serum. These “flow-through” antibodies are saved, and the phospho- and pan-specific antibodies are then eluted from the phosphopeptide affinity column.
The eluted phospho- and pan-specific antibodies are then applied to the dephosphopeptide affinity column. Only the pan-specific antibody binds to this column because the pan-specific peptide sequence but not the phosphopeptide is present. The phospho-specific antibody does not bind to the column and will be in the flow-through (see Figure 3). While the flow-through contains the desired phospho-specific antibodies and is saved, the pan-specific antibodies can be eluted and saved if they are present.