Custom Antibody Services - Characterization

production | purification | characterization

Characterization of the Antibodies: The final stage of custom 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.

western blots

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 1).

figure 1

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 2, 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.

figure 2

immunohistochemistry

The ultimate test: Now we turn to the ultimate test of the antibody: immunohistochemistry. As illustrated in Figure 3, 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 scientist's protein of interest.

figure 3

production | purification | characterization

PhosphoSolutions

Why Go Anywhere Else? As this summary indicates, we devote a great deal of care to the production of phospho-specific antibodies for each individual research scientist's protein of interest. Find out more about our scientists and how to contact us.