A comparison of reagents for detecting and quantitating proteins in solution—Table 9.1 | Thermo Fisher Scientific - MX

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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)	470/570	Quasi-linear from 0.5 to 4 µg in a 200 µL assay volume, with a sample volume of 1–20 µL	Binds 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)	470/570	10 ng/mL to 10 µg/mL	Binds 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)	450/550	10 ng/mL to 150 µg/mL	Reacts 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 450/618	50 µg/mL to 5 mg/mL, with a sample volume of 1 µL	Binds 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 (Coomassie brilliant blue)	595	1 µg/mL to 1.5 mg/mL	Directly 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 (bicinchoninic acid)	562	0.5 µg/mL to 1.2 mg/mL	Cu²⁺ 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 (biuret reagent plus Folin–Ciocalteu reagent)	750	1 µg/mL to 1.5 mg/mL	Cu²⁺ 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 (F2332, F20261)	390/475	0.3 µg/mL to 13 µg/mL	Reacts 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 (o-phthaldialdehyde) (P2331MP)	340/455	0.2 µg/mL to 25 µg/mL	Reacts 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	280	10 µg/mL to 50 µg/mL or 50 µg/mL to 2 mg/mL	Peptide 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.

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