Custom Antibody Services - Characterization

Western blotting illustrates the importance of sequential affinity columns to separate the phosphospecific antibody from the non-phosphospecific and pan antibodies in the serum.

Specificity in Western Blotting

The composite Western blot below in Figure 1 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. First, there are no cross-reacting bands labeled by the antibody in the rat brain homogenate. Second, phospho-specificity 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.


Synapsin phosphospecificity


Now we turn to immunohistochemistry. Many of our antibodies have been tested in IHC. Figure 2 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).


Synapsin IHC

Figure 3 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).

Tyrosine Hydroxylase IHC

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We will be with you through the entire process of making and testing your custom polyclonal antibody.


The crucial first step in the development of custom-made phosphospecific antibodies is the selection and design of the immunizing peptide.


The only way to isolate the desired phosphospecific antibody is through sequential phospho and dephospho affinity chromatography.

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