The quantification of proteins is one of the most common and basic analytical methods in biochemistry. At present, there are several methods commonly used: nitrogen determination method, ultraviolet absorption method, Lowry method (Folin-phenol reagent method), Bradford method (Coomassie brilliant blue method), BCA method, etc.:
The nitrogen determination method is more complicated, but more accurate, the protein determined by the nitrogen determination method is often used as the standard protein of other methods.
Ultraviolet absorption method is simple, sensitive, fast, does not consume the sample, the sample can still be recycled after the determination. The disadvantages are poor accuracy and large interfering substances. When using the standard curve method to determine the protein content, there is a certain amount of error for those proteins that differ greatly from the tyrosine and tryptophan content of the standard protein.
The Lowry method is a sensitive detection method developed earlier, with a minimum sensitivity of 5 micrograms and usually a measurement range of 20-250 micrograms. The advantages are high sensitivity, the disadvantage is that it takes a long time, and it is necessary to precisely control the operation time. The standard curve is not a strictly linear form, and the specificity is poor, and there are many interfering substances.
The Bradford method is a method with higher sensitivity. The advantages are: high sensitivity, about 4 times higher than the lowry method, and a minimum detection of 1-5 micrograms; the assay is rapid, simple, and stable in staining; there are few interfering substances. The exact point is that there are large deviations for different protein determinations; there are still interferences such as SDS, detergents, Triton x100, etc.; the standard curve is slightly non-linear.