Preparation of monospecific antibodies against isoform 2 of translation elongation factor 1A (eEF1A2)

Abstract

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.

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.…”

mRNAs coding for A1 and A2 isoforms of translation factor eEF1A demonstrate different half-lives while A1 and A2 proteins are similarly stable in MCF7 cells

“…Unfortunately, these data did not give a definite answer, even in the case the same cell line was used in different studies (Table). To clarify the issue we measured half-lives of eEF1A1 and eEF1A2 directly, using cycloheximide-treated MCF-7 cells and two kinds of antibodies, the first recognizing both A1 and A2 isoforms and the second being exclusively specific for A2 [17]. As the amount of A1 in MCF7 cells is much larger (by orders) than A2 we consider the former antibodies response reflects mostly the A1 amount.…”