How does Bicinchoninic acid assay work?
The principle of the bicinchoninic acid (BCA) protein assay1 relies on the formation of a Cu2+-protein complex under alkaline conditions, followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the amount of protein present. BCA assays are routinely performed at 37ºC.
What interferes with Bradford assay?
Basic conditions and detergents, such as SDS, can interfere with the dye’s ability to bind to the protein through its side chains. However, there are some detergent-compatible Bradford reagents.
What factors affect BCA assay?
Bicinchoninic Acid Assay (BCA) Method The BCA reagent is stable under alkaline conditions, so it can be included in the biuret alkaline copper solution. The development of color in the BCA assay depends on time, temperature, and pH.
What is the purpose of bicinchoninic acid?
Bicinchoninic acid is used to analyze to monitor cuprous ion presence due to its purple coloration via a biuret reactionIn this assay, two molecules of bicinchoninic acid chelate a single Cu+ ion, forming a purple water-soluble complex that strongly absorbs light at 562 nm.
How might you control for interference in a protein assay?
The effects of such interferences can be ameliorated by strategies such as reducing the interfering substances through dialysis, gel filtration or if the protein concentration is high enough, by diluting the sample.
What are the 2 reagents used in BCA?
Reagents. Reagent A: 1 gm sodium bicinchoninate (BCA), 2 gm sodium carbonate, 0.16 gm sodium tartrate, 0.4 gm NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water.
How does SDS interfere with Bradford assay?
When proteins that carry residue of SDS are used as samples in the Bradford assay, the Coomassie dye is either kept from binding due to the bondage of SDS to proteins, or the SDS associates with the green form of the dye, shifting the equilibrium and overrepresenting the absorption at 595 nm regardless of true protein …
Why do detergents interfere with Bradford assay?
Additionally, free amino acids and small peptides (<3 kDa) do not interfere with the assay. If a quick Bradford protein assay kit with a ready-to-use Bradford reagent is used, detergents that interfere will need to be at a lower concentration in the sample than in the standard assay due to the high sample-to-dye ratio.
Does SDS interfere with BCA assay?
The 2-mercaptoethanol will interfere with the BCA assay, and the SDS will interfere with the Bradford assay.
Why is BSA used in BCA assay?
BSA is used because of its stability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
Why is BSA used in biuret test?
An aqueous solution of bovine serum albumin (BSA) is commonly used as a standard in testing because of its stability in testing, low cost, and is readily available as a byproduct from bovine blood.
What are protein assays used for?
Purpose of Protein Assays The purpose of the protein assay is to determine the amount or concentration of a specific protein or an array of different proteins in a sample. Isolating and detecting protein is used for many clinical and research processes.