Freimoser:Protocols:ProteinQuantification

Absorbance assays

Absorbance at 280 nm

• Range: 20 micrograms to 3 mg
• Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
• Accuracy: Fair
• Convenience: Excellent, if equipment available
• Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets

Absorbance at 205 nm

• Range: Roughly 1 to 100 micrograms
• Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
• Accuracy: Fair
• Convenience: Very good
• Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets

Extinction Coefficient

• Range: 20 micrograms to 3 mg
• Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
• Accuracy: ~2% (very good)
• Convenience: Very good
• Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets

Colorimetric assays

Modified Lowry

• Range: 2 to 100 micrograms
• Volume: 1 ml (scale up for larger cuvettes)
• Accuracy: Good
• Convenience: Fair
• Major interfering agents: Strong acids, ammonium sulfate

Biuret

• Range: 1 to 10 mg
• Volume: 5 ml (scale down for smaller cuvettes)
• Accuracy: Good
• Convenience: Good
• Major interfering agents: Ammonium salts

Bradford assay

• Range: 1 to 20 micrograms (micro assay); 20 to 200micrograms (macro assay)
• Volume: 1 ml (micro); 5.5 ml (macro)
• Accuracy: Good
• Convenience: Excellent
• Major interfering agents: None

Bicinchoninic Acid (Smith)

• Range: 0.2 to 50 micrograms
• Volume: 1 ml (scale up for larger cuvettes)
• Accuracy: Good
• Convenience: Good
• Major interfering agents: Strong acids, ammonium sulfate, lipids

Amido Black method

• Range: 2 to 24 micrograms
• Volume: 2 ml
• Accuracy: Good
• Convenience: Poor
• Major interfering agents: None reported

Colloidal Gold

• Range: 20 to 640 nanograms
• Volume: 1 ml (scale up for larger cuvettes)
• Accuracy: Fair
• Convenience: Poor
• Major interfering agents: Strong bases