Lesson 3.6. Quantitative Protein Analysis

Biuret Test, Bradford Assay, Lowry Assay, and Bicinchoninic Acid (BCA) Test

most common method to determine protein concentration

Principle of Quantitative Protein Analysis

the protein sample is made to react with a compound producing a visible colored product which can be analyzed using spectrophotometer

standard calibration curve

A _______________________ of known protein sample will be utilized to compute for the amount of proteins in the sample using the absorbance with the application of Beer-Lamberts law.

UV Spectrophotometry Method

• Proteins will absorb ultraviolet light at 280 nm.

• This is due to the tyrosine and tryptophan residues in the protein.

• Quantitation is done by comparing the absorbance of the test solution with a known standard.

The method is accurate, simple and highly sensitive up to microgram quantities

Advantage of UV Spectrophotometry Method

Biuret Method

• Cupric ions chelate (complex formation) with peptide bonds of proteins in alkaline medium produce a pink or violet color.

• The intensity of the color is proportional to the number of peptide bonds.

• The color is then compared with a standard protein solution treated with the Biuret reagent, and estimated colorimetrically @540 nm

Advantage of Biuret Method

simple one-step process, and is the most widely used method for plasma protein estimations

Disadvantage of Biuret Method

The sensitivity of the method is less and is unsuitable for estimation of proteins in milligram or microgram quantities.

Bradford Method

• A simple and one of the most commonly used assay for total protein concentration.

• It is based on the proportional binding of Coomassie dye to proteins. The more proteins present, more dye will bind.

• It is a colorimetric assay. The color intensity of the solution depends on the concentration of protein

Principle of Bradford Method

In an acidic medium, proteins bind to Coomassie dye due to hydrophobic and electrostatic interactions

465 to 595nm

Λmax of dye of Bradford Reagent

blue

In Bradford Method, the Red solution turns to _______ solution (stable up to 1 hr)

Principle of Biuret

Light blue to Pink-violet or violet Complex of Cu

Principle of Bradford

Red dye to Coomassie blue; Binding with Coomassie dye

Wavelength of Biuret

540 nm

Wavelength of Bradford

595 nm

Accuracy of Biuret

Less accurate

Accuracy of Bradford

For general use, fairly accurate

Sensitivity of Biuret

Less sensitive; requires higher Detects up to 0.5 to 2.5 mg

Sensitivity of Bradford

Sensitive Detects up to 1 to 20 ug

Compatibility with reagents of Biuret

Incompatible to ammonium salts and reducing agents like dithiothreitol

Compatibility with reagents of Bradford

Compatible with most salts, solvents, buffers, reducing substances and chelating agents. Incompatible with detergents