Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

Authors

  • P.V. Ershov Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • L.A. Kaluzhskiy Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • E.O. Yablokov Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia
  • A.S. Ivanov Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia

DOI:

https://doi.org/10.18097/BMCRM00112

Keywords:

FITC; dye labeling; cytochrome c; interactomics; capillary gel electrophoresis; surface plasmon resonance (SPR)

Abstract

The technology of dye-labeled proteins has many fields of application, especially in interactomics. The aim of this work was to adapt protocol of conjugation of low molecular weight (12 – 15 kDа) heme-containing proteins with fluorescein isothiocyanate, isomer I, (FITC) for subsequent protein-protein interaction studies. We have monitored the quality of FITC-labeling of the target protein and comparative assessment of its binding capacity. Using the cytochrome C (Mw 12 kDа) as an example, it has been shown that using the three step method approach including conventional spectrophotometry, capillary gel electrophoresis and SPR analysis it is possible to assess: (i) the capability of the FITC-labeled target protein to interact with its protein partner and protein material from tissue lysates, (ii) the fact of dye conjugation with the protein, and (iii) the quality of purification for final protein preparation from unreacted free dye molecules

References

  1. Florinskaya, A.V., Ershov, P.V., Mezentsev, Y.V., Kaluzhskiy, L.A., Yablokov, E.O., Buneeva, O.A., Zgoda, V.G., Medvedev, A.E., Ivanov, A.S. (2018) The analysis of participation of individual proteins in the protein interactome formation. Biomeditsinskaya Khimiya, 64(2), 169–174. DOI
  2. Ershov, P.V., Mezentsev, Y.V., Yablokov, E.O., Kaluzhskiy, L.A., Vakhrushev, I.V., Gnedenko, O.V., Florinskaya, A.V., Gilep, A.A., Usanov, S.A., Yarygin, K.N., Ivanov, A. S. (2019) A Method of Lysate Preparation to Improve the Isolation Efficiency of Protein Partners for Target Proteins Encoded by the Genes of Human Chromosome 18. Biomed. Chem. Res. Methods, 2(1), e00090. DOI
  3. Ershov, P.V., Mezentsev Y.V., Kopylov, A.T., Yablokov, E.O., Svirid, A.V., Lushchyk, A.Ya., Kaluzhskiy, L.A., Gilep, A.A., Usanov, S.A., Medvedev, A.E. and Ivanov, A.S. (2019) Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome. Biology (Basel), 8(2), 49. DOI
  4. Ng, S., Smith, M.B., Smith, H.T., Millett, F. (1977) Effect of modification of individualcytochrome c lysines on the reaction with cytochrome b5. Biochemistry, 16(23), 4975-4978. DOI
  5. Arrell, M.S., Kálmán, F. (2016) Estimation of protein concentration at high sensitivityusing SDS-capillary gel electrophoresis-laser induced fluorescence detection with 3-(2-furoyl)quinoline-2-carboxaldehyde protein labeling. Electrophoresis, 37(22), 2913-2921. DOI
  6. Ershov, P.V., Mezentsev, Y.V., Yablokov, E.O., Kaluzhskiy, L.A., Florinskaya, A.V., Gnedenko, O.V., Zgoda, V.G., Vakhrushev, I.V., Raeva, O.S., Yarygin, K.N., Gilep, A. A., Usanov, S. A., Medvedev, A. E., Ivanov, A. S. (2018) Direct Molecular Fishing of Protein Partners for Proteins Encoded by Genes of Human Chromosome 18 in HepG2 Cell Lysate. Russ. J. Bioorg. Chem., 44, 759–776. DOI

Published

2019-12-12

How to Cite

Ershov, P., Kaluzhskiy, L., Yablokov, E., & Ivanov, A. (2019). Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor. Biomedical Chemistry: Research and Methods, 2(4), e00112. https://doi.org/10.18097/BMCRM00112

Issue

Section

EXPERIMENTAL RESEARCH