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Production of affinity purified antipeptide antibodies to survivin for structural and functional studies of the protein

Abstract

Tumor-associated protein survivin is a bifunctional protein that can participate either in the regulation of cell division or in the inhibition of apoptosis depending on its location in the cell and its structural state. The aim of the present work was to obtain monospecific antibodies to survivin targeting various sites in the protein and suitable for the study of its structural and functional features. The ability of the antibodies to detect survivin in tumor cells and mammary tumor tissues has been assessed. Antibodies targeting the (1–22) and (95–105) fragments of the protein have been shown to have the highest specific activity. Antibodies targeting the (1–22) site preferentially bound to survivin-containing complex (presumably a dimer of survivin) in immunoblotting, whereas antibodies targeting the (95–105) site detected only the monomeric form of the protein. Analysis of malignant mammary tumor samples showed that monomeric survivin was present only in tumor tissue samples, whereas survivin-containing complex was expressed both in tumor tissues and in the tissues adjacent to the tumor. Antibodies targeting the (1–22) site were shown to preferentially detect survivin localized in the cell nuclei (and involved in the regulation of mitosis) in immunocytochemistry, while those targeting the (95–105) site stained the nucleoplasm and cytoplasm at all stages of the cell cycle. Therefore, the antibodies obtained can serve as a useful tool for structural and functional research on survivin.

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Abbreviations

AB:

antibodies

ELISA:

enzyme-linked immunosorbent assay

IFA:

incomplete Freund’s adjuvant

CFA:

Freund’s complete adjuvant

BIR:

highly conserved domain characteristic of all members of the apoptosis inhibitor protein family

CPC:

chromosomal passenger complex

DAB:

3,3′-diaminobenzidine

DAPI:

4′,6-diamidino-2-phenylindole dihydrochloride

EDT:

1,2-ethanedithiol

FITC:

fluorescein isothiocyanate

Fmoc:

9-fluorenylmethoxycarbonyl

KLH:

keyhole limpet hemocyanin

OVA:

ovalbumin

PBS:

phosphate buffered saline

pH 7.4:

containing 2.7 mM KCl

140 mM NaCl:

8.1 mM Na2HPO4, and 1.5 mM KH2PO4

RecSurv:

recombinant survivin

TBS-T:

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

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Correspondence to E. V. Akhidova.

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Original Russian Text © E.V. Akhidova, T.D. Volkova, D.O. Koroev, I.Yu. Yakupov, M.V. Kalintseva, L.E. Zavalishina, A.P. Kaplun, O.O. Zharskaya, O.V. Zatsepina, O.M. Volpina, 2013, published in Bioorganicheskaya Khimiya, 2013, Vol. 39, No. 3, pp. 326–337.

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Akhidova, E.V., Volkova, T.D., Koroev, D.O. et al. Production of affinity purified antipeptide antibodies to survivin for structural and functional studies of the protein. Russ J Bioorg Chem 39, 288–298 (2013). https://doi.org/10.1134/S1068162013030023

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  • DOI: https://doi.org/10.1134/S1068162013030023

Keywords

  • survivin
  • synthetic peptides
  • antibodies
  • survivin-containing complex