Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling.

Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.

Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux.

The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.