2 STRATEGIES TO PREPARE NEURAL CORTICAL CYTOSKELETON COMPONENTS FOR THE GENERATION OF MONOCLONAL-ANTIBODIES

Like most other cells, neurons possess a spectrin/actin based network closely associated with the inner side of the cell membrane, the corti cal cytoskeleton. This structure serves many diverse functions during axonal outgrowth. In the growth cone, the cortical cytoskeleton is inv olved in surface shaping, modulation of integral membrane proteins, an d signal transduction. We developed two strategies to prepare material enriched for neural cortical cytoskeleton. The first strategy combine d the isolation of a membrane/cortical cytoskeleton fraction by densit y gradient centrifugation with an enzymatic degradation of cell surfac e proteins. The second strategy is based on the attachment and crossli nking of single cells to beads, allowing for the removal of the cell c ontents by cell disruption; only membrane/cortical cytoskeleton patche s are retained on tbe beads. Both strategies made use of the intimate association of the cortical cytoskeleton with the cell membrane, permi tting the removal of cytoplasm, organelles and cytoplasmic cytoskeleto n while retaining tbe cortical cytoskeleton. Monoclonal antibodies gen erated using both preparations as immunization material were screened for recognition of intracellular structures in axons and growth cones of retinal ganglion cells in culture. A quantitative specification of the antibodies is presented and six antibodies are characterized in im munolabelings and Western blot analysis.