Selective Partitioning of NCAM isoforms with Triton X100

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Selective Partitioning of N-CAM isoforms with
Triton X-100

• James E. Haley, Ph.D.
• College of Mt. St. Vincent , N.Y.

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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

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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

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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

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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)

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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

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A Actin
VVimentin
G GFAP
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