Biomedical Chemistry: Research and Methods 2019, 2(4), e00112

Quality Control of FITC-Labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensorg

P.V. Ershov, L.A. Kaluzhskiy, E.O. Yablokov*, A.S. Ivanov

Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119121 Russia; *e-mail: evgeyablokov1988@mail.ru

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

DOI:10.18097/BMCRM00112

The whole version of this paper is available in Russian.

29 conjugates of methylene blue and four chemical structures, including derivatives of carbazole, tetrahydrocarbazole, substituted indoles and γ-carboline, combined with a 1-oxopropylene spacer have been studied as channel blockers of the NMDA receptor (binding site of MK-801) by using four QSAR methods (multiple linear regression, random forest, support vector machine, Gaussian process) and molecular docking. QSAR models have satisfactory characteristics. The analysis of regression models at the statistical level revealed an important role of the hydrogen bond in the complex formation. This was also confirmed by the study of modeled by docking complexes. It was found that the increase in the inhibitory activity of the part of compounds could be attributed to appearance of additional H bonds between the ligands and the receptor.
Figure 1. SDS-CGE-UV electrophoregrams of unlabeled and FITC-labeled cytochrome c forms as well as FITC preparation itself. For comparative analysis, identical amounts (150 μg) of unlabeled and FITC-labeled cytochrome c forms were used. A sample containing 20 μg free FITC was used for analysis.
Figure 2. SPR immobilization level (n = 2) of the various forms of cytochrome c (unlabeled and FITC-labeled ones) via standard amino coupling protocol (A); the binding levels of unlabeled and FITC-labeled forms of cytochrome c (50 μM) injected as analyte (n = 5) with covalently immobilized CXXC1 protein on the optical chip (B).
Figure 3. SPR binding levels (n = 4) of protein material from different rat liver tissue lysate preparations with different forms of covalently immobilized cytochrome c: unlabeled one (A) and FITC-labeled one (B).

FUNDING

Capillary electrophoresis study was supported by the Russian Foundation for Basic Research (RFBR) (project No 18-04-00071).

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