The key difference between BCA and Bradford assay is that BCA assay is time-consuming and less accurate, whereas Bradford assay is quick and accurate.
BCA and Bradford assay are two assay methods of protein concentration determination. These two assays have different principles of assay and differences in their accuracies.
CONTENTS
1. Overview and Key Difference
2. What is BCA Assay
3. What is Bradford Assay
4. BCA vs Bradford Assay in Tabular Form
5. Summary – BCA vs Bradford Assay
What is BCA Assay?
BCA assay or bicinchoninic acid assay is a biochemical assay useful in determining the total concentration of protein in a solution. This method is also named as Smith assay after its inventor, Paul K. Smith. This analytical method shows similarities to Lowry protein assay, Bradford protein assay or biuret reagent. We can observe the total protein concentration in a given sample through a colour change of the sample solution, which goes from green to purple. This colour change occurs in proportion to the protein content. Thereafter we can use a colourimetric technique to analyse the colour intensity and protein content in the sample.
BCA Method of Protein Estimation
When considering the mechanism of BCA assay, the typical BCA solution contains a highly alkaline solution with a pH of nearly about 11.25, and the ingredients in this solution include bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and copper(II) sulfate pentahydrate. This assay primarily depends on two chemical reactions: the reduction of copper(II) to copper(I) by the peptide bonds in protein and chelation of two molecules of bicinchoninic acid with copper(I) ion, resulting in a purple coloured complex that can absorb light at 562 nm wavelength. In the first reaction, the amount of copper(II) reduced by peptide bonds is proportional to the amount of protein in the sample.
What is Bradford Assay?
Bradford assay is a spectroscopic analytical method useful in measuring the concentration of protein in a solution. This method was developed by Marion M. Bradford in 1976, and it is a comparatively quick and accurate method.
Color Reaction of Protein and Bradford Reagent
We can use this method to determine the concentration of the protein amount in a sample. The chemical reaction that takes place in the sample during this assay is solely dependent on the amino acid composition of the protein in the sample.
Bradford Method of Protein Estimation
When considering the method of this assay, it is a colourimetric protein assay that is based on the absorbance shift of the dye named Coomassie Brilliant Blue G-250. Generally, this dye exists in three forms as anionic (blue) form, neutral (green) form and cationic (red) form. Therefore, under acidic conditions, the dye changes its colour from red to blue. This is because of the binding of the protein with the dye. In contrast, if there is no protein to bind with the dye, the colour change does not appear, the solution gets a red-brown colour. This dye is capable of forming a strong, noncovalent complex with the carboxyl group of the protein through Van der Waal forces and electrostatic attractions.
What is the Difference Between BCA and Bradford Assay?
BCA and Bradford assay are two assay methods of protein concentration determination. These two assays have different principles of assay and differences in their accuracies. The key difference between BCA and Bradford assay is that BCA assay is time-consuming and less accurate, whereas Bradford assay is quick and accurate.
The following infographic summarizes the differences between BCA and Bradford assay in tabular form.
Summary – BCA vs Bradford Assay
BCA assay or bicinchoninic acid assay is a biochemical assay useful in determining the total concentration of protein in a solution. Bradford assay is a spectroscopic analytical method useful in measuring the concentration of protein in a solution. The key difference between BCA and Bradford assay is that BCA is time-consuming and less accurate, whereas Bradford assay is quick and accurate.
Reference:
1. “Bradford Protein Assay.” Experimental Biosciences, Rice University.
Image Courtesy:
1. “Bradford protein assay” By Helito – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “BSA Standard Curve” By Alexieogo – Own work (CC BY-SA 4.0) via Commons Wikimedia