Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
Labeling of molecules such as antibodies or enzymes with biotin is done by use of derivatized biotin molecules. For conjugation purpose, biotin-hydrazide and biotin-N-hydroxysuccinimide (with or without spacer arms, e.g. aminocaproic acid) are employed which couple via carboxyl groups or primary amine groups. In the latter case, the number of amines available for conjugtaion will vary among proteins, Thus, reaction conditions being optimal for one protein may not be optimal for another protein. The appropriate extent of labeling can be controlled in a set of preliminary experiments in which a series of different biotin concentrations are used to select the conjugate with the best staining results and the lowest background.