Protein AssaysBelow is a list of assays for the determination of protein concentration in a solution. This list includes the sensitivity range, volume/amount of sample needed, subjective comments on accuracy and convenience, and major interfering agents. Procedural details, equipment requirements, and references are outlined in the individual assay documents.
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.
General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).
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
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
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
Range: 2 to 100 micrograms
Volume: 1 ml (scale up for larger cuvettes)
Accuracy: Good
Convenience: Fair
Major interfering agents: Strong acids, ammonium sulfate
Range: 1 to 10 mg
Volume: 5 ml (scale down for smaller cuvettes)
Accuracy: Good
Convenience: Good
Major interfering agents: Ammonium salts
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
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