Hello colleagues, I am also getting the same issue while performing egg albumin and bovine serum albumin assay using my plant extract and dexamethasone at 660 nm. Results always go in negative value. But after heating the reaction mixture at 70 degrees for 5 min absorbance at 280 nm is good. But all the 05 concentrations (0.833, 1.667, 3.334, 6.668, 13.334, and 26.67 µg/ml) of dexamethasone gave results as 98.72%, 98.96%, 99.12%, 99.20%, 99.24%, 99.22%, respectively. Similarly, with plant extract, the results were better at 280 nm, but at 660 nm last three higher concentrations (400, 800 and 1600 µg/ml) of plant extract resulted in negative values. So, I don't know if results with 280 nm should be considerable or not in terms of egg albumin assay. I saw many articles stating the results with 280 nm but most of the articles go with 660 nm. So, I want to optimize this methodology, then please suggest which wavelength would be most acceptable and what's the mechanism with such wavelength in terms of protein denaturation.

I would appreciate it if someone could clarify and help me to understand it clearly.

I really appreciate any help you can provide.