ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
Biotechnologia Acta Т. 17, No. 3, 2024
P. 59-65, Bibliography 16, Engl.
UDC: 577.218+616-006
doi: https://doi.org/10.15407/biotech17.03.059
Full text: (PDF, in English)
GENERATION AND CHARACTERIZATION OF POLYCLONAL ANTIBODIES SPECIFIC TO HUMAN ESTROGEN RECEPTOR ERα
Institute of Molecular Biology and Genetics of the Natonal Acvademy of Scienes of Ukraine, Kyiv
Aim. The purpose of the study was to generate and characterize anti-hERα polyclonal antibodies for elucidation of functional relationships between isoforms of estrogen receptor ERα and isoforms of ribosomal
protein S6 kinase — S6K1.
Methods. cDNA cloning. Expression of recombinant proteins in bacterial system. Affinity purification of His-tag fused recombinant proteins using Ni-NTA chromatography from bacterial lysates. Generation of polyclonal sera by mice immunization. Western blot analysis and immunoprecipitation.
Results. cDNA coding for full length hERα was cloned into expression vector pET28a in frame with His-tag sequence. Recombinant hERα-His protein was expressed in E.Coli and purified by Ni-NTA chromatography.
Purified hERα-His was used as antigen for mice immunization and generation of polyclonal antibodies. Specificity of polyclonal antibodies was analyzed by Western blot and immunoprecipitation of hERα from MCf-7 cell lysates.
Conclusions. Generated anti-hERα polyclonal antibodies are of conformational type since specifically recognized hERα only in immunoprecipitation but not in Western blot. Created polyclonal antibodies a suitable for detection and analysis of hERα protein complexes.
Key words: human estrogen receptor α (ERα), cloning, expression of recombinant proteins, antibodies, immunoprecipitation.
© Palladin Institute of Biochemistry of National Academy of Sciences of Ukraine, 2024
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