At right is an example of a peptide design for a serine phosphorylation site on tyrosine hydroxylase. The rationale for the design of this peptide is as follows:

Truncated N-Terminal sequence: The truncated N-terminal sequence eliminates cross reactivity with homologous proteins that were identified in a Blast search.

Use of a Short Sequence: A very short sequence forces the phosphoseryl residue into the epitope recognized by the antibody.

Amidation of the 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 an N-Terminal Cysteinyl Residue: Lastly, an N-terminal cysteinyl residue was added for conjugation to the carrier protein and coupling to the affinity column.

Phospho-peptide

• Conjugated to KLH for use as antigen for immunizations
• ELISA screens for reactivity
• Positive sequential affinity purification

Non-phospho peptide

• ELISA screens for phosphospecificity
• Negative sequential affinity purification

Sequential affinity columns separate the phosphospecific antibody from the non-phosphospecific and pan antibodies in the serum.

After immunization with the phospho peptide, the rabbit’s immune response can produce three types of antibodies. As seen in the figure at right, the first is the phosphospecific antibody that is desired. However, antibodies that are specific for the dephospho form of the peptide and the total protein (pan) can also be generated. The dephospho antibody can be generated against the peptide if phosphatases in the rabbit dephosphorylate the peptide conjugate that was injected. 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 regardless of its phosphorylation state. The only way to isolate the desired phosphospecific antibody is through sequential phospho- and dephosphoaffinity chromatography.

Sequential affinity chromatography begins by applying the rabbit serum to the phosphopeptide affinity column (left). Two of the three types of antibodies described above will bind to this column: the phosphospecific antibody (blue) will bind because the phosphopeptide is present on this column. Pan-specific antibodies (red) will also bind because they recognize the peptide regardless of the phosphoryl group. The dephospho form of the peptide isn't present, so dephospho antibody (green) is the only one of the three that will not bind. It passes through the column in the flow-through, along with 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. We call this fraction the Pre-Non-Phospho fraction (PNP).

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 it recognizes this peptide as well. The phosphospecific antibody does not bind to the column and will be in the flow-through. This is the Affinity-Purified fraction (AP). While the flow-through contains the desired phosphospecific antibodies and is saved, the pan-specific antibodies can be eluted and saved if they are present. This final fraction is the Non-Phospho-Elute fraction (NPE).

For phosphospecific antibodies, ELISAs are performed to test the antibody's reactivity against both the phospho and non-phospho peptides. An effective phosphospecific antibody must show high affinity for the phosphopeptide while demonstrating near-zero cross-reactivity with the non-phosphopeptide.

Samples are tested for initial serum screening and then from every step in the purification process: from the serum to the column flow-through to the eluted antibody to the column wash.

Western blotting illustrates the importance of sequential affinity columns to separate the phosphospecific antibody from the non-phosphospecific and pan antibodies in the serum. After receiving your antibody from us, test the provided fractions in Western blot using appropriate lysates to determine the phosphospecificity.

An example of the importance of specificity in Western blotting:

The composite Western blot below shows the characteristic doublet of our synapsin pan antibody as well as three of our phospho synapsin antibodies. Phosphospecificity is definitively demonstrated by comparing the signal in untreated rat brain homogenate (left) to the same homogenate, but that has been treated with lambda and alkaline phosphatase prior to being run on the gel (right). Treatment with phosphatase had no effect on the pan antibody’s signal, but completely eliminated the immunolabeling of the phospho antibodies.

Immunohistochemistry can be used to validate phosphospecific antibodies by highlighting specificity in real tissue, especially by comparing staining in serial sections before and after phosphatase treatment. If the signal disappears after dephosphorylation, you know the antibody is genuinely phosphospecific.

IHC allows for confirmation of expected activation patterns, like comparing stimulated versus control cells. Unlike Western blots that rely on denatured proteins, IHC proves your antibody works in native tissue with endogenous expression levels and preserved cellular architecture.

Many of our antibodies have been tested in IHC. The figure at right shows staining of cultured neurons with anti-synapsin pan antibody in green (top), and of C57 mouse striatal cells with anti-synapsin Ser549 (lower).