Witch wavelength is best for measuring protein in the Biuret test? I came across different variants ranging from 540-565nm, with the reagent consisting of the same chemicals. I tried measuring at all of the wavelengths but there are big differences, really significant for my results, and I wonder which is the most appropriate.
Also the Biuret test gives me like 2-times more protein than I detect with the Bradford assay. This cannot be due to smaller peptides, I am pretty sure I don’t have any degraded protein in the sample. Any clue why I get this?
Yes the two assays were preformed independently with separate standard curves of BSA.
Well our proteins are more soluble at alkaline conditions and since the Bradford reagent is acidic and the Biuret alkaline, the solubility is probably better in the Biuret. You think that the Bradford assay wouldn’t detect insoluble (due to pH) proteins present in the sample? If so this could be it.
The biuret method is based on detection of peptide bonds and is used to assay the proteins in a liquid which contains many proteins (> 5 g / l). The absorption spectrum of the complex formed is wide and the choice of the wavelength is not very important. The Bradford assay uses coomassie blue that reacts in an alkaline medium with different amino acids. It is generally used to assay small amounts of proteins. If I understand, you have standardized with an albumin solution. The proteins that are present in your solution may be very different from that protein. The intensity of the coloration of the two techniques can be influenced by the amino acid composition of your proteins. Think also to verify that the Beer-Lambert law is respected with both techniques in the area of concentration of your protein mixture
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