Characterization of novel peptide-specific antibodies against the translation elongation factor eEF1A2 and their application for cancer research

Abstract

Aim.We intend to characterize the new peptide-specific antibodies against the isoform 2 of translation elongation factor 1A (eEF1A2) and determine its presence in the postoperative samples of human breast, lung and stomach tumor tissues. Methods. The analysis of antibody specificity was performed by enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry. Immunoblotting and immunohistochemistry were used for the determination of the eEF1A2 in the human tumor samples, as well as in the samples of normal tissues surrounding tumors. Results. The antibodies obtained against the eEF1A2 specifically recognized this protein in the cell extracts and histological sections and did not cross-react with the elongation factor 1A isoform 1. eEF1A2 was revealed in the postoperative samples of breast, lung and stomach tumors as well as in the putative normal tissues surrounding tumors. Conclusions. The antibodies obtained against eEF1A2 are highly specific for the antigen and can be used for the immunological studies of tumors.

Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues

“…Affinity purification of antibodies against glutarylated proteins. IgG fractions from immunized rabbit sera were prepared by ammonium sulfate fractionation followed by separation from other proteins on DEAE Blue agarose (DEAE Blue Affi-gel, «Bio-Rad Laboratories») [25,26] in 20 mM PB, pH 7.2.…”

“…In our previous work, we used sequential immunization cycles with the replacement of the carrier protein at the same hapten to obtain anti-peptide antibodies that selectively interacted with one of the highly identical isoforms of the eukaryotic translation elongation factor 1A, 1A2 (eEF1A2) [26]. A similar approach was used to obtain antibodies to glutarylated proteins in the current work.…”

Protein isoforms. Origin, structure and functions.

“…We suggest that the eEF1A1 and eEF1A2 isoforms may participate in different processes in cell. The physical basis for this is their different spatial organization, an ability to form dimers, different lipophilic properties [65][66][67], different number and level of posttranslational modifications [68][69][70]. The X-ray structure is only known for eEF1A2 [71].…”

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“…We suggest that the eEF1A1 and eEF1A2 isoforms may participate in different processes in cell. The physical basis for this is their different spatial organization, an ability to form dimers, different lipophilic properties [65][66][67], different number and level of posttranslational modifications [68-70]. The X-ray structure is only known for eEF1A2 [71].…”