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  • Title: [Construction and expression of prokaryotic expression vector of GAP-43 and preparation of monoclonal antibody against GAP-43].
    Author: Duan XL, Huang WJ, Wang BR, Song JF, Jin WL, Guo X, Ju G.
    Journal: Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi; 2003 Sep; 19(5):480-2. PubMed ID: 15169663.
    Abstract:
    AIM: To express the growth-associated protein-43 (GAP-43)in prokaryotic cells and prepare monoclonal antibody( mAb)against GAP-43. METHODS: Full length sequence of GAP-43 gene was amplified from the plasmid containing pGAP-43cDNA and was cloned into the expression vector pGEX-4T-I.GST-GAP-43 fusion protein was expressed in E. coil under IPTG induction. Expressed fusion protein was purified by glutathine agarose chromagraphy, Using purified protein as an immunogen, mAb against GAP-43 was prepared. RESULTS: The recombinant plasmid containing the target gene was constructed successfully. The fusion protein was expressed in E. coli in soluble form. The titer of anti-GAP-43mAb in ascites was 1: 10'. The Ig subclass and subtype of the mAb was IgG2a and K type, respectively. Specificity of the mAb was confirmed by ELISA, Western blot and immunofluorescence technique. CONCLUSION: The anti-GAP-43 mAb obtained has strong specificity and high titer, which provides an useful reagent for further studying the function of GAP-43 in nervous system.
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