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Absorbance assays

• absorbance at 280 nm
• absorbance at 205 nm
• extinction coefficient

Colorimetric assays

• set up an assay
• spectrophotometry
• modified Lowry
• biuret
• Bradford
• Bicinchoninic Acid (Smith)

Protein Assays

The criteria for choice of a protein assay are usually based on convenience, availability of protein for assay, presence or absence of interfering agents, and need for accuracy. For example, the Lowry method is very sensitive but is a two step procedure that requires a minimum of 40 minutes incubation time. The Bradford assay is more sensitive and can be read within 5 minutes, however proteins with low arginine content will be underestimated. Generally, estimates are more accurate for complex mixtures of proteins. Estimates of concentration of pure proteins can be very inaccurate depending on the principle of the assay, unless the same pure protein is used as a standard. Criteria will be discussed in the individual documents.

Because different proteins have different amino acid compositions, the sensitivity of colorimetric assays to individual proteins may vary widely. The most reprodicible results are obtained with standards composed of a mixture of proteins that is as similar as possible to the unknown. For most purposes, a relative amount is good enough, but the standard used should be reported. For example, the Bradford assay is much more sensitive to bovine serum albumin (BSA) than to immunoglobulin G (IgG), so that with IgG the investigator is likely to overestimate the amount of protein in a sample. With BSA the investigator is likely to underestimate the amount.

General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

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