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List of methods Direct absorbance measurement absorbance at 280 nm absorbance at 205 nm extinction coefficient Colorimetric assays set up an assay spectrophotometry modified Lowry biuret Bradford Bicinchoninic Acid (Smith)	Estimating protein concentration Below is a list of methods for determining 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 method are usually based on convenience, availability of protein, 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 method, 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). Direct measurement of absorbance 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
	Copyright and Intended Use Visitors: to ensure that your message is not mistaken for SPAM, please include the acronym "Bios211" in the subject line of e-mail communications Created by David R. Caprette (caprette@rice.edu), Rice University 7 Sep 95 Updated 12 Jun 15