The BCA protein assay is used to quantify total protein in a biological sample. BCA stands for Bicinchoninic acid, which is the key reagent used to produce a colored product. The purple colored product is analyzed in reference to a standard curve in order to quantify protein concentration. It is important to measure protein concentration after performing a protein extraction or purification, and prior to any type of labeling procedure. The protein concentration after extraction or purification may provide information about a biochemical pathway or a disease state. All commercially available proteins are accompanied by a product information sheet that has the results of a protein quantification method. This is particular important in antibody validation. It is important to know the protein concentration prior to any labeling step so you can ensure that the stoichiometric ratio between label and protein is optimal for clean and efficient labeling. It is equally important to know how much protein you are working with when designing biosensors so that you can define limits of detection and instrument sensitivity.
How the BCA assay quantifies protein concentration
The BCA assay requires two chemical reactions in order to produce the purple colored product that can be measured by spectrophotometry at 562 nm. The most common instrument used to measure this product is a plate reader, because a standard protein concentration curve is needed to produce a truly quantitative result. The BCA protein assay is best used to detect protein concentrations between 20 and 2000 µg/mL.
The two steps of the BCA protein assay and how the color is formed
- Cu2+ is added to the sample and is reduced to Cu1+ by protein. This step must be performed in basic solution with sodium potassium tartrate. This step is sometimes referred to as the biuret reaction and it forms light blue color that is very faint and difficult to quantify on its own. The three amino acids most involved in the reaction are cysteine, tyrosine, and tryptophan. The peptide backbone also contributes to the reaction, which makes this assay different from the coomassie protein detection method.
- Bicinchoninic acid is added to the sample to react with the Cu1+ produced by the protein. This second step produces a purple reaction product that is much brighter than the blue color produced in step 1. Two molecules of BCA react with each Cu1+. By adding this second reaction step the BCA assay is 100x more sensitive than if it were only composed of detecting the product from step 1.
Other protein assays and detection methods:
There are a variety of other protein detection methods used to quantify protein concentration. Perhaps the simplest and least invasive methods is simply to use a spectrophotometer to measure absorbance of 280 nm light. The side chains of aromatic amino acids are responsible for absorbing this UV light. Therefore, this is an easy way to quickly estimate protein concentration.
Another method is to label primary amines with ninhydrin or o-phthalaldehyde and measure the colorimetric or fluorescent product.
The Coomassie or Bradford assay measures the direct binding of dye to protein. This produces a colorimetric or fluorescent product.
The BCA protein assay and modified Lowry assay are both copper chelation mechanisms.
The choice of protein assay used to quantify protein concentration will depend on the sample itself and any downstream applications. Will it be important not to introduce any extra reagents to the sample? Are there other components to the sample that could interfere with the protein detection method? This is particularly important when considering the BCA protein assay because anything other than the protein in the sample that reacts with cuprous ions could produce inaccurate results. Whatever method you choose it is important to understand the mechanism of the assay to ensure that you have an accurate readout of protein concentration.
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