5 METHODS TO QUANTIFY PROTEINS

The precise quantification of the proteins in a sample is essential for studying them in an infinity of research areas.

While the quantification of a specific protein can be carried out by means of tests such as Western Blot or ELISA , by mass spectrometry (MS) or by other methods such as those based on nanoparticles, there are tests that allow quantifying the concentration of total protein present in a shows.

This last case is the one we focus on this week, compiling a brief description of the 5 main methods to quantify total proteins .

1.- BICINCHONINIC ACID (BCA)
  • Concentration range : 20-2000 ug / ml
  • Features :
    • Developed in 1985
    • Colorimetric test where the absorbance will be proportional to the concentration of protein present in the sample
    • Two-step reaction:
      • Formation of protein-copper ion complexes
      • Formation of a Cu-BCA chelate resulting in an intense purple coloration that absorbs at 562nm
    • It is a recommended method in the case of samples containing> 5% detergents and / or denaturing agents such as urea or guanidinium chloride.
  • Limitations :
    • Samples containing substances that interact with copper, such as ammonia, like EDTA, reducing sugars, or lipids, may interfere with this assay.
2.- ULTRAVIOLET (UV) ABSORPTION
  • Concentration range : 0.1-100 ug / ml
  • Features :
    • This method, by measuring the characteristic absorption of tryptophan and tyrosine at 280nm, estimates the amount of protein present in the sample.
  • Limitations :
    • It is incompatible with protein extraction methods that use detergents and / or denaturing agents.
    • It is not a specific method for proteins, since other compounds that could be in the sample also absorb at 280nm (such as alcohols or nucleic acids, among others)
3.- BRADFORD OR COOMASIE BLUE TEST
  • Concentration range : 20-2000 ug / ml
  • Features :
    • Method described in 1976
    • It is characterized by being a quick, simple and compatible method with reducing agents used to stabilize proteins in solution.
    • The negatively charged Coomasie blue stain binds to positively charged proteins.
    • It mainly binds to arginine, tryptophan, tyrosine, histidine and phenylalanine residues.
    • The solution stain is red and absorbs at 465nm, while binding to the basic amino acids of a protein it turns blue and absorbs at 595nm.
    • The absorbance measurement is compared with the values ​​of a standard curve to determine the protein concentration of the sample.
  • Limitations :
    • Not valid for detecting proteins of <3kDa
    • It is incompatible with detergents such as SDS or Triton X-100
4.- LOWRY’S ESSAY
  • Concentration range : 10-1000 ug / ml
  • Features :
    • One of the most widely used methods to quantify proteins was developed in 1951
    • It is a very sensitive and precise test
    • Like the BCA assay, a two-step reaction occurs:
      • Formation of Cu-N complexes (present in the protein)
      • The tyrosine and tryptophan complexes react with the Folin-Ciocalteau rectifier, giving rise to a greenish-blue color that absorbs between 650 and 750nm.
    • The absorbance measurement is compared with the values ​​of a standard curve to determine the protein concentration of the sample.
  • Limitations :
    • It is incompatible with certain chemical reagents in common use such as Tris, EDTA, DDT, 2-mercaptoethanol, carbohydrates, etc.
5.- CBQCA (3- (4-CARBOXYBENZOYL) QUINOLINE-2-CARBOXYDEHYDE) ASSAY
  • Concentration range : 100 ng – 1500 ug / ml
  • Features :
    • It is a highly sensitive fluorogenic agent that is used for the detection of primary amines in proteins.
    • The intensity of the fluorescence emitted will be directly proportional to the concentration of protein present in the sample.
    • Does not interact with lipid compounds
  • Limitations :
    • The result depends on the number of certain amino acids present in the protein.