Wednesday, 5 May 2021

Bradford Assay for Protein Quantification

 The Bradford test or Bradford assay is a technique used for Protein Quantification. It is one among many colorimetric assays used to determine the total protein concentration in a solution.

• The bradford method is based on a dye named coomassie brilliant blue(G-250).
• In an acidic solution this reagent is protonated and has a reddish or brownish color.
• In the presence of protein coomassie brilliant blue stably binds to them. The dye protein complex quickly takes a blue color.
• An acidic environment is required for this reaction.
• The complex formation results in a shifted absorption maximum of the dye.
• The protein solution can be measured in a spectrophotometer at 595 nm.
• In the absence of protein there is no change in color. The absorption maximum does not shift.

Interaction between Coomassie Brilliant Blue and the Proteins

In an acidic environment coomassie brilliant blue disrupts the protein structure. That exposes the side chains of the amino acid. 
  The sulfonic acid groups of the dye interact with the positive amino groups of the protein found in basic amino acid residues present in lysine, arginine and histidine.
  This causes a spectral shift from red/brown form of the dye ( absorbance at 465 nm) to the blue form of the dye (absorbance at 595 nm).

Determination of the protein concentration

  The spectrophotometer measures the optical density at 595 nm.
Protocol -
• First the pure solution without any protein content is measured.                                    

•  To calculate the protein concentration of an unknown sample, we need a reference solution where the protein concentration is already known. This is classified as the standard.

• In most cases the protein BSA(Bovine serum albumin) is used for this.

• Different dilutions of this standard are made.

• The optical density is measured for 1 μg/μl and higher concentrations such as 2 μg/μl and 4 μg/μl.

• That generates a linear curve in which the optical density increases proportionally with protein concentration. 

• Now the sample with the unknown protein content is measured. The spectrophotometer assigns the optical density and with extrapolation, we can calculate the protein concentration, which will be 3 μg/μl as per figure. 


Advantages 

• the coomassie dye interacts quickly with the proteins. The assay can determine protein concentrations in a very short time.
• It is also easy to use since it just requires the bradford reagent and a spectrophotometer.
• The assay can be performed at room temperature in comparison to other protein quantification techniques.
• The bradford method is extremely sensitive, it can even detect protein concentrations of 1 microgram per milliliter.

Disadvantages

• One of the main disadvantage is interference with detergents such as SDS.
• The coomassie dye reacts mainly with the basic amino acids the assay faces another limitation it is dependent on the protein's amino acid composition.

The bradford assay is just one among many protein quantification techniques.

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