FNR cluster conversions were followed under pseudo-first-order conditions (with oxygen in excess) by measuring absorbance changes at 420 nm.
In general, I R is determined by hybridization conditions, immobilization conditions, and surface chemistry of the slide, but independent from the spotting concentration if the amount of allelic sequences in the spotting solution is in a great excess over the absorbance capacity of the slide surface.
The samples were then reduced using excess sodium hydrosulfite, and absorbance was again measured between 510 and 630 nm.
To remove the excess reagent interference in the absorbance measurement of the reaction product, 0.2 mL of concentrated HCl was added.
To remove the excess reagent interference in the absorbance measurement of the reaction product, this excess was acid-hydrolyzed to colorless 2,4-dinitrophenol by adding 0.2 mL of concentrated HCl allowing the measurement of ALD-DNFB derivative which remains stable.
Based on this analysis (Table 1) using excess antigen, we found that absorbance plateaued from 50 μg/ml to 3.125 μg/ml of capture antibody, with the drop occurring using 0.78 μg/ml.
This lack of absorbance suggests all excess ICG-sOSu in the reaction to be associated with either the aggregate species or desired conjugation product, with this late peak primarily composed of impurities originating from the commercial ICG-sOSu (Supporting Information Figure S1).
For Method II, to remove the excess DNFB reagent interference in the absorbance measurement of the reaction product, this excess was acid-hydrolyzed to colorless 2,4-dinitrophenol by adding 0.2 mL of HCl allowing the measurement of PG-DNFB derivative which remains stable.
The concentration of the excess Dox was determined by the absorbance measurement at 480 nm using a calibration curve (Supplementary Figure S4).
Addition of excess H2O2 to MitoB gave a UV absorbance spectrum identical to that of MitoP.
