- Make Au-HRP solutions at mole ratios of Au:HRP from 60 to 150.
- Analyze the concentration of HRP stock solution with UV-vis.
- The molar absorption coefficient of HRP at 403 nm is 1.02×105 M-1 cm-1.
- Dialyze fractions of ADA made on x, x, and x.
- Ten solutions at Au-HRP mole ratios of 60, 70, 80, 90, 100, 110, 120, 130, 140, and 150 were made.
- Please refer to Dhea Patel's entry for theoretical and actual calculations of stock solutions, and volumes of stock solutions and deionized H2O added to each solution.
- Note that 130, 140, 150 Au-HRP solutions were recalculated to account for a new stock solution at different concentration of HRP.
- The concentration of the new HRP stock solution was calculated from its absorbance at 403 nm and its molar absorptivity coefficient.
ADA Activity Assay with Adenosine
- Frac 1,3 of ADA, made on x, was used. Its concentration was 10.35 μM, as determined by Bradford assay.
- It was diluted to 1 ng/μL, then to 0.1 ng/μL using phosphate buffer, prepared on x.
Initial concentration of ADA: 10.35 μM in 1 mL of solution
(10.35×10-6)(0.001 mL) = 1.035×10-8 mol ADA
(1.035×10-8 mol)(42000 g/mol) = 0.0004347 g ADA/mL
(0.0004347 g ADA/mL)(1 mL/1000 μL)(109 ng/1 g) = 434.7 ng/μL
(434.7 ng/μL)(V1) = (1 ng/μL)(1000 μL)
V1 = 2.3 μL
(1 ng/μL)(V1) = (0.1 ng/μL)(1000 μL)
V1 = 100 μL
R&D Systems ADA Activity Assay