Working with Antibodies

Correspondingly, two principal immunohistochemistry methods are employed direct and indirect. The direct method is a straightforward one-step process that creates a direct reaction between the antigen and the labeled antibody. The indirect method requires the use of two antibodies a primary unlabeled antibody and a secondary labeled antibody. 

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One of the best ways to ensure retention of activity in protein molecules is to avoid doing chemistry at the active center. The active center is that portion of the protein where ligand, antigen, or substrate binding occurs. In simpler terms, the active center (or active site) is that part that has specific interaction with another substance (Means and Feeney, 1971). For the preparation of enzyme derivatives, it is important to protect the site of catalysis where conversion of substrate to product happens. For instance, when working with antibody molecules, it is crucial to stay away from the two antigen binding sites. 

Work with antibodies to receptors has provided important information about the mechanism of action of prolactin, and should provide further insights in future. It... 

Dissolve the antibody to be modified at a concentration of l-10mg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 7.2, containing 10 mM EDTA. High levels of EDTA often are required to stop completely metal-catalyzed oxidation of sulfhydryl groups when working with serum proteins—especially polyclonal antibodies purified from antisera. Presumably, carry-over of iron from partially hemolyzed blood is the contaminating culprit. 

Washes and dilutions wash sections in PBS or TBS for 2x3 min. PBS can be used for all washes and dilutions. When working with AP-labeled secondary antibodies, TBS must be used. 

Note Do not use sodium azide when working with HRP-conjugated antibodies. 

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