Why do protein solutions have to be alkalised in biuret test?
This is probably to prevent precipitation of copper hydroxide (see Itzhaki &amp; Gill, 1964 - they suggest adding dilute copper sulfate slowly to the NaOH solution to avoid this). If you have the protein already alkalized and ready to react you'll get the color reaction before precipitate forms.

Commercial premixed solutions, like @canadianer mentioned in a comment, have tartrate present to prevent this (Geiger &amp; Bessman, 1972 mention this).

It doesn't seem like it's strictly necessary to add NaOH first, but that's probably the most reliable way to do the assay without using an additional stabilizing agent.



Itzhaki, R. F., &amp; Gill, D. M. (1964). A micro-biuret method for estimating proteins. Analytical biochemistry, 9(4), 401-410.

Geiger, P. J., &amp; Bessman, S. P. (1972). Protein determination by Lowry's method in the presence of sulfhydryl reagents. Analytical biochemistry, 49(2), 467-473.