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

Колесанова, Е. Ф.; Farafonova, T. E.; Aleshina, E. Yu.; Pyndyk, N. V.; Veremieva, M. V.; Novosylna, O. V.; Kovalenko, M. I.; Shalak, V. F.; Negrutskii, B. S.

doi:10.1134/s1990750813010083

Cited by 4 publications

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“…Synthesis of antigenic peptide KSDPPQEAAQFTSQ, its purification and conjugation to keyhole limpet hemocyanin and ovalbumin were performed as described in [26]. Conjugates were characterized by determining the number of peptide copies per molecule of conjugate by MALDI-TOF mass spectrometry [26].…”

Section: Methodsmentioning

confidence: 99%

“…Conjugates were characterized by determining the number of peptide copies per molecule of conjugate by MALDI-TOF mass spectrometry [26].…”

Section: Methodsmentioning

confidence: 99%

“…This procedure was repeated three times in order to replace the previous sample buffer solution with PBS. After the last round of concentration the antibody titers and protein concentrations were measured as described in [26]. The fractions with the highest titer were diluted with glycerol up to 50 % (v/v) and stored at -20°C.…”

Section: Methodsmentioning

confidence: 99%

“…Characterization of antipeptide antibodies against eEF1À2 by immunohistochemistry and immunoblotting. Recently, we have shown that the immunization of mice with KSDPPQEAAQFT SQ peptide conjugated to BSA led to the generation of antibodies that specifically recognized eEF1A2, but not eEF1A1 [26]. However, mouse immunization did not allow obtaining the amount of antibodies sufficient for multiple histochemical trials; hence, a large-scale anti-peptide antibody preparation via goat immunization was started.…”

Section: Methodsmentioning

confidence: 99%

“…Currently, a monospecific antibody preparation is the only appropriate tool that can be made. Recently, we have shown that mouse immunization with one of the eEF1A2 fragments conjugated to bovine serum albumin (BSA) led to the generation of antibodies that specifically interacted with purified eEF1A2, but not with eEF1A1 [26]. In this paper we characterize the peptide-specific antibodies obtained after immunization of goat with the abovementioned peptide conjugated to hemocyanin and ovalbumin.…”

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confidence: 99%

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

et al. 2014

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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.

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Section: Methodsmentioning

confidence: 99%

“…Conjugates were characterized by determining the number of peptide copies per molecule of conjugate by MALDI-TOF mass spectrometry [26].…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

et al. 2014

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

et al. 2021

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The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

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Anti-Peptide Antibodies in the Era of Monoclonals: Application in Diagnostics and Research

2022

Biotechnology: State of the Art and Perspectives

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

Cited by 4 publications

References 20 publications

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

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

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

Anti-Peptide Antibodies in the Era of Monoclonals: Application in Diagnostics and Research

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