Biotinylated antibodies, mixture

To measure the degree of biotinylation of the sample, add an aliquot of biotinylated antibody of known concentration to 1 mL of avidin-HABA complex For example, add 0.05-0.1 mL of biotinylated antibody at 1 mg/mL to 1 mL of avidin-HABA mixture Bring the volume to 1 10 mL, if necessary, incubate for 10 min, and measure the decrease in absorbance at 500 nm. 

Mixture of D-penicillamine (pen, Fig. 22) and copper(II) chloride forms a complex of the formula [Cu(I)8Cu(II)6pen12]5-. The Ri value of this complex at 300 MHz and 23°C is 0.586 mM 1 s-1 [178]. The carboxylate groups on the cluster have been shown to react with aziridines such as XAMA-7 (Fig. 23) which in turn can be reacted with biotin [179]. The copper clusters can then be attached to antibodies or other biotinylated proteins via an avidin linker molecule. 

Fig. 9. Aspartate aminotransferase immunoreactivity in glutamic acid decarboxylase (GAD)-immunoreactive neuronal processes in the cerebral cortex. sAAT but not mAAT is colocalized with GAD in fine, probably axonal processes (arrows). Rat sections were double-immunostained by incubation with a mixture of anti-sAAT or mAAT rabbit serum and anti-GAD sheep serum, then with biotinylated anti-rabbit IgG donkey antibody, and finally with Texas Red-conjugated avidin and fluorescein-labeled anti-sheep IgG donkey antibody. The photographs in each row were taken at the same site under different excitations. Asterisks in (a) and (o ) indicate the unlabeled cell body of a pyramidal neuron.

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