Production and characterization of an anti-peptide antibody specific for the growth-associated protein, GAP-43.
In an attempt to raise specific anti-GAP-43 antibodies, a C-terminal tetradecapeptide was synthesized based on the predicted rat GAP-43 amino acid sequence using the F-moc polyamide solid phase procedure. The synthesized carboxy-terminal peptide was purified by reverse phase HPLC, conjugated to bovine serum albumin (BSA) and used as an immunogen. Polyclonal antipeptide antibodies raised in rabbits were affinity purified and their specificity for GAP-43 tested by Western blotting. Brain and spinal cord homogenates and GAP-43 enriched synaptosomal membrane fractions were analysed either by SDS-PAGE or reverse phase HPLC. The anti-GAP-43 antibodies detected a major immunoreactive band at 43 kDa (B-50), and a minor immunoreactive band at 38 kDa (B-60) together with an additional protein-band at 68 kDa, which was related to the peptide carrier, BSA. Immunohistochemical studies using these carboxy-terminal antipeptide antibodies revealed a widespread distribution of GAP-43 immunoreactivity throughout the adult rat brain and spinal cord, in a pattern generally consistent with earlier histochemical studies. It is concluded that the C-terminal GAP-43 specific tetradecapeptide is a potent immunogen and provides a convenient tool for the production of sequence specific anti-GAP-43 antibodies.
Wiese
U H
UH
MRC Group, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge, U.K.
Emson
P C
PC
Sheppard
R C
RC
D000375:Aging; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000166:cytology* / Q000254:growth & development; D001923:Brain Chemistry; D002846:Chromatography, Affinity; D019922:GAP-43 Protein; D007136:Immunoglobulins / Q000302:isolation & purification; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010750:Phosphoproteins / Q000276:immunology; D051381:Rats; D011919:Rats, Inbred Strains; D013116:Spinal Cord / Q000737:chemistry* / Q000254:growth & development; D013570:Synaptic Membranes / Q000737:chemistry; D013574:Synaptosomes / Q000737:chemistry
D019922:GAP-43 Protein; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins