Determination of the amount of protein in an unknown sample is known as protein assay. Different methods are appropriate for the protein assay depending on the amount and purity of protein and the required accuracy of the assay. The most simple and direct way of protein assay involves the measurement of absorbance of the solution at 280 nm, which gives the absorbance in accordance with the proportion of aromatic amino acid content. However, there are several colorimetric and reagent-based methods for the determination of protein concentration, and each of them has its own advantages and disadvantages.
Some of the protein assay methods are:
UV-Visible absorbance at 280nm
In this process, protein concentration is determined by measuring absorbance at 280 nm. The sensitivity of the assay depends on the number of aromatic rings containing amino acid residues.
- Samples are not destroyed
- It is a quick method and doesn’t require any assay reagent
- Absorbance is affected by the pH and ionic strength
- Extremely sensitive to contamination from buffers, biological materials, and salts
Biuret methods of protein assay
Bicinchoninic acid (BCA)assay
The biuret reaction, in which the protein backbone chelates Cu2+ ions and reduces them to Cu+ ions. The Cu+ ions react with BCA in the second step to form a purple-colored product that absorbs at 562 nm.
- The working reagent is very stable
- Compatible with various detergents
- Differences in amino acid composition have less of an impact on the BCA assay.
- The reaction doesn’t complete when it is carried out at room temperature.
- The reducing agent present at the buffer may interfere.
Folin- Lowery assay
This assay is dependent on the presence of aromatic amino acids present in proteins. In this assay, first, the complex between the cupper and peptide bond of aromatic amino acids is formed, which then reacts with the phosphomolybdic/phosphotungstic acid to give an intense color that absorbs at 650-750 nm.
- It can measure at any wavelength between 650 nm and 750 nm with little loss of color intensity.
- The color varies with the different proteins.
- It gives stable results.
- Takes time to perform an assay.
- The assay is photosensitive so it required consistent illumination during the assay.
Colorimetric dye-based methods
The Bradford assay
Bradford’s method is based on a blue dye (Coomassie Brilliant Blue) that binds to free amino groups in the side chain of amino acids, especially Lysine.
- It is fast and easy to perform.
- The assay can be carried out at room temperature.
- This is compatible with salts, solvents, and buffer solutions.
- This method is incompatible with the detergents.
- The response to different proteins can vary greatly, so the choice of standard is critical.
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The Kjeldahl method of protein assay
This method involves estimating the nitrogen content of a sample after it has been converted into ammonia via heating with sulfuric acid, steam distillation, and back titration with sodium hydroxide.
- It is reproducible.
- It is generally used for the determination of protein concentration in food.
- A relatively high amount of protein sample is required for this assay.
- It also measures the non-protein nitrogen along with the nitrogen in protein.
Fluorescent method of protein assay
In this method, the fluorescence intensity of the protein sample solution is measured, and the concentration is determined by using a calibration curve obtained from the fluorescence emission curve of the standard pure protein samples. The intrinsic fluorescence of the aromatic amino acids tryptophan, tyrosine, and/or phenylalanine is measured to determine the protein concentration.
- It has high sensitivity and requires less amount of samples.
- The assay can be adapted for automated handling applications.
- It requires specialized equipment.
Criteria for choosing the protein assay methods
- Compatibility with the sample and its components
- Sample volume required
- Desired speed
- Accuracy and sensitivity of the assay
- Protein-to-protein consistency
- The speed and convenience of testing a large number of samples
- A spectrophotometer for the measurement of absorbance.