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Preparation of antibodies capable of recognizing the Glu/Lys amino acid substitution at position 129 of the survivin sequence

Abstract

Survivin is an oncofetal protein involved in the inhibition of apoptosis and the regulation of cell division. The functions of survivin are determined by its structural state (monomer or dimer). Owing to the natural polymorphism, either the Glu or the Lys residue can be at position 129 of the amino acid sequence of survivin. Lys has the capability for acetylation, and only the protein containing the acetylated residue Lys129 tends to form a dimer. Thus, antibodies recognizing the amino acid substitution Glu129Lys can serve as a tool in the structural and functional investigations of survivin. For preparing the target antibodies, survivin fragments containing residue 129 were synthesized, rabbits were immunized with synthetic peptides, and the antibodies were purified by affinity chromatography on Sepharose conjugated with the corresponding peptides. It was shown by ELISA and immunoblotting that the affinity-purified antibodies are capable of recognizing the amino acid substitution Glu129Lys in the sequence of recombinant and endogenous survivin.

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Abbreviations

Ab:

antibody

CFA:

complete Freund’s adjuvant

Fmoc:

9-fluorenylmethoxycarbonyl

IFA:

incomplete Freund’s adjuvant

KLH:

keyhole limpet hemocyanin

PBS:

phosphatebuffered saline containing 2.7 mM KCl, 140 mM NaCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.7

RecSurv:

recombinant survivin

TBS-T:

buffer containing 20 mM Tris-HCl, 137 mM NaCl, and 0.1% Tween 20, pH 7.6

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Correspondence to T. D. Volkova.

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Original Russian Text © T.D. Volkova, E.V. Askarova, D.O. Koroev, A.V. Kamynina, M.P. Filatova, I.Yu. Yakupov, O.M. Volpina, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 4, pp. 443–450.

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Volkova, T.D., Askarova, E.V., Koroev, D.O. et al. Preparation of antibodies capable of recognizing the Glu/Lys amino acid substitution at position 129 of the survivin sequence. Russ J Bioorg Chem 40, 410–416 (2014). https://doi.org/10.1134/S1068162014040141

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  • DOI: https://doi.org/10.1134/S1068162014040141

Keywords

  • survivin
  • polymorphism E129K
  • synthetic peptides
  • antibodies