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Bradford Assay

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Bradford Assay

General Aim of Bradford Assay Simulation

To measure the concentration of proteins in a solution by colorimetric analysis using Bradford assay test.

 

Bradford Method

Protein concentration measurement according to Bradford assay method.

Learning Objectives (ILOs)

  • By the end of the Bradford assay experiment, student will:

  • Identify the amino acids by using  Bradford test for protein measures.

  • Describe the color change that occurs when proteins combine with Coomassie dye under acidic conditions.

  • Perform serial dilutions of standard.

  • Illustrate the correct standard curve equation for an example BSA standard.

  • Interpret the correct Bradford assay calibration curve equation when given example data.

  • Calculate the protein concentration of an example.

  • Recall the substance commonly used as standards in the assay and the device used to measure the color of the samples.

Theoretical Background / Context

Bradford assay explained:

  • Bradford’s assay is Colorimetric. It’s based on color changes that occur when the protein in the sample interacts with a specific reagent causing color changes that can be measured using a spectrophotometer. 
  • Bradford solution reagent contains the coomassie blue dye. The cationic (unbound) form is green /red with an absorption maximum at 465 nm. The anionic bound form of the dye, held together by hydrophobic interactions, has an absorption maximum at 595 nm. 
  • Thus; the increase of absorbance from 465 to 595 nm is directly proportional to the amount (concentration) of protein bound to the dye.

Principle of Work

  •  The Bradford assay is based on the binding of the basic amino acids of a protein to a dye present in the Bradford reagent (e.g. Coomassie brilliant blue). 
  • dmjnThis leads to a shift in the absorbance maximum of the dye from 465 to 595. 
  • Bradford assay standard curve protocol depends on creating a standard curve of protein solutions with known concentrations, the protein concentration of unknown samples can be plotted on the standard dilution curve and calculated through Bradford assay analysis. 

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