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The biuret reaction is also the basis of the bicinchoninic acid assay (BCA) developed in 1985 by Smith. The cuprous complex from the biuret reaction reacts with the sodium salt of bicinchoninic acid to produce a deep blue color with an absorbance detected at 562 nm. The method detects proteins in the range of 0.2–50μg. The BCA reagent is stable under alkali conditions and so can be included in the biuret alkaline copper solution. The assay can be run at room temperature or sensitivity can be increased at 60 °C. The assay is compatible with detergents, giving it an advantage over both the Lowry and Coomassie dye assays.
Figure 1. Determination of Protein Concentration by the Smith Bicinchoninic Acid Method.
1. Spectrophotometer absorbing in the visible range and 1 mL plastic or glass cuvettes.
2. Reagent A: 1 g sodium bicinchoninate, 2 g Na2CO3, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 g NaHCO3 made up to 100 mL with water. Adjust the pH to 11.25 with NaOH.
3. Reagent B: 0.4 g CuSO4 · 5H2O in 10 mL water. Reagents A and B are stable.
4. Mix 100 volumes of reagent A with 2 volumes reagent B for a working solution. The solution is stable for a week at room temperature.
1. Set the spectrophotometer to read at 562 nm, allowing 15 min for the instrument to equilibrate.
2. Prepare a BSA protein standard at 1 mg/mL concentration in duplicate. Dilute the protein standard in a volume of 20μL to give five concentrations over a range of 10–50μg protein. Each dilution of each of the two standards should be done in duplicate to produce four readings per protein concentration.
3. Add 20μL of protein sample to 1 mL of working solution and incubate for 30 min at 37 or 60 °C for increased sensitivity. Cool the sample and place in a 1 mL cuvette and read the absorbance.
1. Advantages of the Smith bicinchoninic acid assay: (a) The method is less complicated than the Lowry assay; (b) the method is more sensitive than the Lowry assay; (c) the method has less protein: protein variability than the Bradford assay; (d) the volume of reagents can be reduced and a micro-assay can be performed in 96-well plates; (e) detergents are compatible with the assay (up to 5 %); and (f) the method can be automated.
2. Disadvantages of the Smith bicinchoninic acid assay: (a) Reducing reagents (e.g., ascorbic acid) that reduce copper interfere with the assay; (b) chelating agents (e.g., EDTA) that chelate copper interfere with the assay; (c) the method is destructive to proteins; that is, once the protein sample has reacted with the copper, the protein cannot be used for other assays.
3. It is advisable to dissolve the protein standard in duplicate. If a protein standard is weighed or solubilized incorrectly in a single preparation, then all protein determinations will be inaccurate.
4. Protein determinations with the copper (BCA) reagents should always be considered as a relative concentration of protein. It is accepted practice to express the relative concentrations of proteins during protein purification steps, provided that all protein concentrations are determined with the same method.
5. The copper (BCA) protein determination methods all produce a nonlinear curve, whilst absorbance at 280 nm produces a straight line as protein concentration increases.
6. The copper (BCA) protein curves all proceed from the origin of the graph, provided that the correct "blank" or "control" has been used.
Reference
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