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Measuring the abundance of proteins in a sample is essential for biological studies and medical diagnostics. For instance, a sudden rise or drop in the concentration of a particular protein after adding a drug suggests a key role in the mechanism of the biological response. In the laboratory, accurate protein concentrations are needed to perform Western blotting and chemical reactions, such as fluorophore conjugations. In the clinic, measuring interleukins or other cytokines in COVID-19 patients using highly sensitive methods such as ELISAs may help predict the severity of the disease (PMID: 32283152).
There are several methods for determining the concentration of protein in samples and reagents. Among them, the BCA, Bradford, and ultraviolet (UV) methods are the most widely used in scientific research. Each method relies on different scientific principles and has its own advantages and disadvantages. For our HumanKine® cytokines and growth factors, we use BCA for its high sensitivity and range. For our antibodies, we previously used the Bradford method; however, we have now transitioned to the UV method using the Nanodrop spectrophotometer due to the added sensitivity and low sample volume requirements.
The BCA method uses a colorimetric reaction between BCA (Bicinchoninic acid), copper (Cu2+) sulfate, and proteins. Under alkaline conditions, the peptide bonds in proteins reduce divalent copper ions to monovalent copper ions. Monovalent copper ions are then chelated by BCA to form a stable complex that absorbs purple light, turning the solution from green to purple. Since the reduction reaction is proportional to protein concentration, the light absorption of the solution at 562nm (purple) by a plate reader or spectrophotometer can then be used to calculate protein concentration.
Wide detection range (20-2000ug/ml).
Simple, quick (~45min) protocol.
Does not require expensive machinery.
The Bradford method, also known as the Coomassie brilliant blue method, is named after its inventor Marion Bradford from the University of Georgia. Coomassie brilliant blue is a triphenylmethane dye that changes color from red in its cationic, unbound form, to blue in its anionic form when bound to protein. When protein is present, the negative sulfonic groups of the red dye molecules donate electrons to positive side chains such as lysine and arginine. This destabilizes the protein, promoting van der Waals interactions and exposing hydrophobic residues with the dye to form a stable complex with the dye in its blue anionic form. The absorption peak of the blue form is 595nm and the optical density of the complex has a linear relationship with the protein content with a certain range.
One-step, <10min assay.
Does not require expensive machinery.
Uses a significant amount of sample.
Reaction is affected by presence of common protein surfactants (e.g., Triton X, SDS).
Not useful for proteins with above- or below-average amount of basic amino acids.
**Note: Coomassie brilliant blue G250 for detecting protein concentration and Coomassie brilliant blue R250 for staining PAGE gel are not the same! G250 quickly reacts with protein but stains the gel slowly, so it is best for protein quantification. R250 reacts slowly with protein, stains the gel faster, and is easy to elute – making it better suited for PAGE gel staining.
The UV method uses an ultraviolet spectrophotometer to estimate protein concentration. It takes advantage of the property that aromatic amino acids (including tryptophan, lysine, and phenylalanine) in protein molecules contain conjugated double bonds which can absorb UV light (maximum absorption peak is at 280nm wavelength). Based on the Beer-Lambert Law, UV absorption can be used to calculate concentration.
Does not require any additional reagents.
Uses a tiny amount of sample.
Assay does not affect protein activity.
There is no perfect solution for the determination of protein concentration, so selecting a detection method that suits your sample best is important.
1. Zhou Yuenan, Wang Zhan, etc. Talking about the quantitative detection method of protein content[J]. Food Research and Development, 2014.
2. Fan Shuangshuang, Li Zhengping. Comparison of several methods for detecting protein concentration[N]. Journal of Chengde Teachers College for Nationalities, 2004
3. Fan Huajie, Li Wenjie. Reasonable application of protein quantitative methods[J]. Chinese International Medical Journal, 2003