What are the limitations of BCA assays?

One significant disadvantage is that the reaction's sensitivity to protein quantification can be affected by the specific amino acids present in the solution. Although the BCA assay is less sensitive to the type of amino acids in general, it is notably influenced by the presence of cysteine, tryptophan, and tyrosine residues. Additionally, the BCA assay involves preparing a working solution by combining the provided reagents, which adds another step to the process. The assay also involves long incubation times, typically ranging from 30 minutes to 2 hours. Another limitation is that reducing agents, acidifiers, reducing sugars, copper chelating agents, lipids, and phospholipids can all interfere with the assay and impact the results. Lastly, to ensure accurate results, the BCA assay relies on a standard curve generated from a known protein sample. Thus, it is essential to assay both the samples and the known protein standards simultaneously, using the same temperature and incubation time.