Recording begins with the spectrophotometer zeroed (blue arrow) while the flow cell is filled with carrier buffer (PBS). Next, absorbance sharply increases when the flow cell is packed with beads. By zeroing the spectrophotometer again (red arrow), the beads’ absorbance is eliminated and a baseline for BIA measurement is established, allowing the capture of antibody (red bar) to be monitored at a desired wavelength. For routine BIA measurements, the spectra are recorded only after beads are packed and rendered “invisible” to spectrophotometer by a second zeroing as shown here. All steps of this protocol are carried out automatically by means of software script.
Making Beads "Invisible"
Sephadex-Protein A beads, Insulin monoclonal Ab, Injected sample
volume 40 μL, 2 μg Ab/μL, carrier PBS, flow rate 1 μL/sec.
3.4.5.
