I'm new to the FRAP assay. When I mixed FeCl3 with TPTZ before adding buffer, I got a dark blue solution instead of a straw-brown solution.
I used the following method:
2 ml of acetate buffer (pH 3.6) + 2 ml TPTZ+ 20 ml of 10 mM ferric chloride solution
(2mL of 40 mM HCl dissolved in 6.25mg TPTZ to prepare TPTZ solution)