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Here 5μl of each activated medium was mixed with 1 ml of 0.5 M ethanolamine–HCl solution (pH 7.5) for the purpose of blocking the NHS or aldehyde (CHO) group. The resultant mixture was then stirred at room temperature for 2 h. After removing the supernatant by centrifugation, the particle was washed twice with 1 ml of phosphate-buffered saline (PBS). In the case of NHS-activated media, the amount of immobilized NHS was calculated according to the absorbance of the supernatant at 280 nm. Human serum solution (1 ml, diluted 5with PBS) was applied to the medium, and the mixture was stirred at room temperature for 1 h. After that, the resultant mixture was centrifuged and the medium was washed seven times with 1 ml of PBS. Subsequently, 100μl of 0.2 M glycine–HCl (pH 2.5) was applied to the medium, and the mixture was stirred at room temperature for 0.5 h. The resultant mixture was centrifuged, and 50ll of supernatant, 50ll of 1.0 M Tris–HCl (pH8.3), and 100ll of Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) were mixed and heated in boiling water for 3 min (acid elution fraction). Furthermore, after washing the particle twice with 0.2 M glycine–HCl (pH 2.5), 100ll of Laemmli sample buffer was applied to the particle and heated in boiling water for 3 min. After that, the resultant mixture was centrifuged and the supernatant was obtained (sodium dodecyl sulfate [SDS] elution fraction). These elution fractions were applied to a 5–15% acrylamide gel (Bio-Rad Laboratories), and SDS–polyacrylamide gel electrophoresis (PAGE) was performed. The gel was stained with a silver staining kit (Silver Stain II Kit, Wako Pure Chemical Industries), and the level of nonspecifically adsorbed proteins was confirmed。 |
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