Custom Rabbit Polyclonal Antibody Design, Purification, and Characterization

Peptide Design

The sequence of the immunizing peptide is crucial to antibody specificity.

The crucial first step in custom antibody development is peptide design. The specificity and utility of the antibody depends critically on the immunizing peptide. As an example of the peptide design process, consider figure below, illustrating the phospho-peptide for serine-40 in tyrosine hydroxylase. The sequence N-terminal and C-terminal to the phospho-serine is shown at the middle of the figure, while the peptide design is shown at the bottom.

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 a 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 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.

Purification - Three Antibody Possibilities

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

After immunization with the phospho peptide, three types of antibodies can be produced in the rabbit’s immune response. As seen in the figure 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 PhosphoSpecific Antibody

The first column we use is the phosphopeptide affinity column (below, left). 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 (below, right). 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.

Antibody Characterization

After purification we characterize the antibody in ELISA. Western blot and IHC or ICC can also be used for additional analysis and visualization.

ELISA

For phosphospecific antibodies, ELISAs are performed against both phospho and non-phospho peptides to determine the antibody's degree of preferential recognition for the phospho peptide. We test a sample from every step in the purification process, from the serum to the column flow through to the eluted antibody to the column wash.

WESTERN BLOT

Western blotting illustrates the importance of sequential affinity columns to separate the phosphospecific antibody from the non-phosphospecific and pan antibodies in the serum. Many times, we have appropriate positive lysate already in-house. If we don't, you can choose to provide us with samples of your lysate or cells so that our team can work with you to characterize your antibody.

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, and demonstrates two very import aspects of antibody specificity.

1. There are no cross-reacting bands labeled by the antibody in the rat brain homogenate.
2. 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

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

The figure below illustrates our pan-TH antibody’s extensive labeling in this photomicrograph of the retina (left). In contrast, a phospho-TH antibody selectively labels only the two amacrine cells in this light-stimulated retina example (right).