& Data Analysis
Protein gel analysis
|List of methods
Direct absorbance measurement
Estimating protein concentrationBelow 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