Table 9.1 A comparison of reagents for detecting and quantitating proteins in solution.

Assay Detection Wavelength(s) (nm) * Sensitivity and Effective Range Mechanism of Action Notes
Quant-iT protein assay (Q33210)

Qubit protein assay (Q33211, Q33212)
470/570Quasi-linear from 0.5 to 4 µg in a 200 µL assay volume, with a sample volume of 1–20 µLBinds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent
  • Extremely fast and easy—just add sample to diluted dye and read fluorescence
  • High sensitivity
  • Little protein-to-protein variation
  • Compatible with salts, solvents, 2-mercaptoethanol, amino acids and DNA, but not detergents
NanoOrange protein quantitation assay (N6666)470/57010 ng/mL to 10 µg/mLBinds to detergent coating on proteins and hydrophobic regions of proteins; the unbound dye is nonfluorescent
  • High sensitivity
  • Little protein-to-protein variation
  • Rapid assay with a simple procedure
  • Compatible with reducing agents, but not detergents
CBQCA protein quantitation assay (C6667)450/55010 ng/mL to 150 µg/mLReacts with primary amine groups on proteins in the presence of cyanide or thiols; the unreacted dye is nonfluorescent
  • High sensitivity
  • Sensitivity depends on the number of amines present
  • Linear over an extended range of protein concentration
  • Compatible with detergents and lipophilic proteins
  • Not compatible with buffers containing amines or thiols
EZQ protein quantitation assay (R33200)280 and 450/61850 µg/mL to 5 mg/mL, with a sample volume of 1 µLBinds electrostatically to basic amino acids, supplemented by additional hydrophobic interactions
  • Ideal for determining protein concentration prior to electrophoresis
  • Solid-phase format designed for high-throughput analysis
  • Little protein-to-protein variation
  • Compatible with detergents, reducing agents, urea and tracking dyes
Bradford assay ref (Coomassie brilliant blue)5951 µg/mL to 1.5 mg/mLDirectly binds specific amino acids and protein tertiary structures; the dye's color changes from brown to blue
  • High protein-to-protein variation
  • Not compatible with detergents
  • Rapid assay
  • Useful when accuracy is not crucial
BCA method ref (bicinchoninic acid)5620.5 µg/mL to 1.2 mg/mLCu2+ is reduced to Cu+ in the presence of proteins at high pH; the BCA reagent chelates Cu+ ions, forming purple-colored complexes
  • Compatible with detergents, chaotropes and organic solvents
  • Not compatible with reducing agents
  • The sample must be read within 10 minutes
Lowry assay ref (biuret reagent plus Folin–Ciocalteu reagent)7501 µg/mL to 1.5 mg/mLCu2+ is reduced to Cu+ in the presence of proteins at high pH; the biuret reagent chelates the Cu+ ion, then the Folin–Ciocalteu reagent enhances the blue color
  • Lengthy procedure with carefully timed steps
  • Not compatible with detergents or reducing agents
Fluorescamine ref (F2332, F20261)390/4750.3 µg/mL to 13 µg/mLReacts with primary amine groups on proteins; unbound dye is nonfluorescent
  • Sensitivity depends on the number of amines present
  • Reagent is unstable
  • Not compatible with amine-containing buffers
OPA ref (o-phthaldialdehyde) (P2331MP)340/4550.2 µg/mL to 25 µg/mLReacts with primary amine groups on proteins in the presence of 2-mercaptoethanol; unbound dye is nonfluorescent
  • Sensitivity depends on the number of amines present
  • Not compatible with amine-containing buffers
  • Low cost
UV absorption ref28010 µg/mL to 50 µg/mL or 50 µg/mL to 2 mg/mLPeptide bond absorption; tryptophan and tyrosine absorption
  • Sensitivity depends on the number of aromatic amino acid residues present
  • Nondestructive
  • Low cost
* Excitation and emission wavelength maxima or absorption wavelength maximum, in nm.
For Research Use Only. Not for use in diagnostic procedures.