Selective Partitioning of N-CAM isoforms with Triton X-100 • James E. Haley, Ph.D. • College of Mt. St. Vincent , N.Y.
Experimental Objectives • Overview of N-CAMs • Culture of Rat Brain Astrocytes • Resolution of Proteins: IEF & 2-D PAGE • Western Blot Analysis of N-CAMs • Results/ Conclusions
Neural- Cell Adhesion Molecules- N-CAMs • Cell surface glycoproteins found on Astrocytes and on Neurons • Variety of molecular weight isoforms: 180, 140, and 120 Kdal • Non-Calcium dependent cell to cell Interactions/ Adhesion
Astrocytes • Major class of glial cells within the central nervous system • Marker Protein- Glial Fibrillary Acidic Protein (GFAP) • Physically supports and protects neurons • Serves to buffer K+ • Functions to Aid in Neurotransmission, especially GABA • Participates in the formation of the Blood-Brain Barrier
Western Blot Analysis • Isoelectric Focusing (IEF) • Second Dimensional Polyacrylamide Gel Electrophoresis • Transfer to Nitrocellulose Paper • Probe with Primary Antibody: Rabbit ant-Rat N-CAMs • Probe with Secondary Antibody: Biotinylated anti-Rabbit IgG • Incubation with Avidin-Peroxidase • Color development with 4-chloronaphthol (4-CN)
Conclusions • High M.W. N-CAMs from Cultured Astrocytes Partitioned into the Triton X-100 Soluble Fraction. • Low M.W. N-CAMs, on the Other Hand Partitioned into the Triton X-100 Insoluble, Cytoskeletal Fraction. • RBC Membranes, a negative Control, showed no detectable N-CAMs • A Whole Cerebellar Extract, a positive control, showed all isoforms of N-CAMs
A =Actin V=Vimentin G = GFAP