Hi everyone,
Is anyone here familiar with the CUPRAC method? I am struggling with creating the standard curve of that method. My procedure is the following: 500ul PBS 1X + 500ul CuSO4 1M + 500ul Neocuproine 1M (I dissolved neocuproine in DI water, adding a small amount of HCl 1M to totally dilute it + a small amount of NaOH 1M to balance the pH back to 6) + 500ul H2O2 at different concentration (0, 0.1, 0.25, 0.5, 1, 2mM). After 30min, I read the peak at 450nm by UV-Vis.
The resulting peaks should be linear and increase as the concentration of H2O2 increases. However, the peaks of H2O2 0.5, 1, and 2M are even smaller than others. I used quartz cuvettes, and carefully wash them after each test. The baseline I used is PBS 1X.
I still don't know what point is wrong with my procedure.
If anyone can help me with that. I appreciate it a lot.
It looks like you did not read original papers on this method. Where did you take the protocol? It's wrong.
An alumni sent this paper to me and said that she follows the procedure in Section 2.7: Article Hyperbaric oxygen-generating hydrogels
CUPRAC method is for antioxidant activity, e.g.
Article The main and modified CUPRAC methods of antioxidant measurement
Your protocol is wrong from chemical point of view.
Find the protocol for H2O2 measurements suitable for your system.
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