RESUMO
Amino acid sequences of eukaryotic translation elongation factor isoform 1 (eEF1A1) and 2 (eEF1A2) were compared and two peptide fragments of eEF1A2 were chosen as linear antigenic determinants for generation of monospecific antipeptide antibodies. Selected peptides were synthesized, conjugated to bovine serum albumin (BSA) and used for mice immunizations. Antibodies, produced against the eEF1A2 fragment 330-343 conjugated to BSA, specifically recognized this isoform in the native and partially denatured states but did not interact with the eEF1A1 isoform. It was shown that these monospecific anti-eEF1A2 antibodies could be employed for eEF1A2 detection both by enzyme-linked immunosorbent assay and by immunoblotting.
Assuntos
Anticorpos/imunologia , Western Blotting/métodos , Fator 1 de Elongação de Peptídeos/análise , Peptídeos/química , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Bovinos , Humanos , Imunização , Fígado/química , Camundongos , Dados de Sequência Molecular , Músculo Esquelético/química , Fator 1 de Elongação de Peptídeos/imunologia , Peptídeos/síntese química , Peptídeos/imunologia , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Soroalbumina Bovina/químicaRESUMO
Peptide RHDSGY that represents the fragment of human beta-amyloid Zn-binding site and its isomers RH(D-Asp)SGY and RH(beta-Asp)SGY have been prepared by solid-phase synthesis and analysed by HPLC and various mass-spectrometric methods. The problem of low yield of peptide RHDSGY and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of side-products as RH(Asp-imide)SGY and RHDSGY (instead of RH(beta-Asp)SGY) was solved via selection of reagents for the removal of Fmoc groups from the growing peptide chain.