Fig 1: Isolation of the BChE–protein complexes by size-exclusion chromatography (SEC) of serum proteins. (A) Chromatogram of human serum proteins separated on the TSK gel 3000 SWXL (Tosoh, Tokyo, Japan). Protein elution was monitored at 280 nm. The first 13 fractions of 200 µL were collected every 12 s in separate tubes. (B) Determination of the BChE activity in the fractions by Ellman’s assay in the collected fractions. The BChE activity is shown as dA412/min in red numbers above corresponding bars. dA412/min stands for change in absorbance at 412 nm per minute. (C) Analysis of the protein complexes in the 13 fractions by native polyacrylamide gel electrophoresis on Mini-PROTEAN 4–15% TGX Stain-Free Gels (Bio-Rad, Hercules, CA, USA). Ten microliters of each fraction was separated under native conditions in TB buffer at 4 °C. (D) SDS-PAGE analysis of the protein complexes in the 13 fractions. Ten microliters of each fraction sample was separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). M—protein marker; 1–13—10 µL of fractions. (E) Western blot analysis of 13 fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA) (1/200 dilution). (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution).
Fig 2: Fractionation of BChE–protein complexes by density iodixanol gradient ultracentrifugation (OptiPrep). (A) The separation procedure is summarized in the flow chart. (B) Picture of the centrifuged tube taken after separation. Positions of fractions are marked on the tube. (C) BChE activity of 10 uL of each fraction was estimated by Ellman’s assay as it has been described in Figure 1B. The BChE activity is shown as dA412/min. (D) Sudan black stained agarose gel electrophoresis profile of separated fractions after electrophoresis. Ellman’s reaction was developed in 100 mM PB buffer (pH 7.4) with a final concentration of 0.5 mM DTNB and 5 mM BTC. S—1 µL of serum; 4–15—1 µL of fractions. (E) SDS-PAGE; 1 µL of the fraction samples were separated on Mini-PROTEAN 4–15% precast TGX Stain-Free gels (Bio-Rad, Hercules, CA, USA). SDS-PAGE and Western blotting were performed according to the method described in Figure 1D. S—1 µL of serum; M—protein marker; 4–15—1 µL of fractions. (F) Western blot analysis of the fractions using rabbit monoclonal anti-ApoB antibody (ab139401) (Abcam, Cambridge, UK) (1/2000 dilution). (G) Western blot analysis of the fractions using rabbit monoclonal anti-ApoA-I antibody (ab52945) (Abcam, Cambridge, UK) (1/2000 dilution). (H) Western blot analysis of the fractions using mouse monoclonal anti-BChE antibody (D-5): sc-377403 Santa Cruz Biotechnology (Santa Cruz, CA, USA)(1/200 dilution). (I) Western blot analysis of the fractions using rabbit monoclonal anti-vitronectin antibody (ab46808) (Abcam, Cambridge, UK) (1/2000 dilution).
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