Here you find a step by step description how PhosphoSolutions produces their highly specific phospho-specific antibodies.
The first step in the development of phospho-specific antibodies is the selection and design of the immunizing peptide. This is a crucial step because the specificity and utility of the phospho-specific antibodies depends critically on the design of the immunizing peptide.
Drs. Andrew Czernik and Dr. Michael Browning, who together have more than three decades of experience in designing and producing phospho-specific antibodies, are responsible for the design of the immunizing peptides at PhosphoSolutions. As an example of this process, the phosphopeptide for serine-40 in tyrosine hydroxylase is [C]RQSpLIEDAR-NH2. The sequence N-terminal and C-terminal to the phospho-serine is EAVTSPRFIGRRQSLIEDARKEREAAA.
The rationale for the design of this peptide is as follows:
Truncated N-Terminal sequence: First, the peptide has a truncated N-terminal sequence. This was done to eliminate cross reactivity with homologous proteins that were identified in a Blast search.
Use of Short Sequence: Second, another feature of the peptide design was the use of a very short sequence which forces the phosphoseryl residue into the epitope recognized by the antibody.
Amidation of C-Terminus: Third, given the very favorable amphipathic character of the peptide, the C-terminus was amidated to better emulate the native tyrosine hydroxylase protein. Such modifications can improve immunoreactivity in western blots and especially in immunohistochemical applications.
Addition of N-Terminal Cysteinyl Residue: Lastly, an N-terminal cysteinyl residue was added for conjugation to the carrier protein and coupling to the affinity column.
Three antibody possibilities: (See figure 1) Multiple affinity chromatography steps are essential in order to produce antibodies that are phospho-specific. This is due to the fact that immunization with a phosphopeptide can generate three different types of antibodies. 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 will bind because the phosphopeptide is present on this column. The pan-specific antibody will also bind because the sequence/conformation 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, as this form of the peptide is not present on the column. These latter antibodies elute from 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 our desired phospho-specific antibodies and is saved, the pan-specific antibodies can be eluted and saved if they are present.
The final stage of phospho-specific antibody production is the characterization of the antibodies. We use differential dot blots for our initial characterization and to establish that the phospho-specificity of the antibody reflects at least two orders of magnitude preference for phosphopeptide over that of the dephosphopeptide. However, western blots are essential to truly establish the specificity of the antibodies for the holoprotein of interest.
Specificity of the antibodies: The first Western blot analysis simply demonstrates that there are no cross-reacting bands labeled by the antibody in a homogenate or cell lysate. Thus, as seen in a Western blot of increasing concentrations of caudate nucleus homogenate using the phospho-tyrosine hydroxylase antibody, only a single band is labeled at the appropriate molecular weight in SDS-PAGE (Figure 4).
The second level of specificity uses western blots of phospho- and dephospho-proteins to demonstrate the selectivity of the phospho-specific antibody. As shown in Figure 5, the panspecific antibody we obtained recognized both the dephosphotyrosine hydroxylase (TH) and the phospho-TH. Most importantly, the phospho-specific antibody recognized only phospho-TH whereas the dephospho-specific antibody reacted selectively with the dephospho-TH.
The ultimate test: Now we turn to the ultimate test of the antibody: immunohistochemistry. As illustrated in Figure 6, the pan-TH antibody shows extensive labeling in this photomicrograph of the retina. In contrast, the phospho-TH selectively labels only the two amacrine cells in this light-stimulated retina example. As this summary indicates, we devote a great deal of care to the production of phospho-specific antibodies for an individual research scientists protein of interest.
PhosphoSolutions Phospho-Specific Antibodies