Measuring Protein Concentration: Overview of Common Protein Assay Methods
Proteins play essential roles in virtually every biological process within living organisms. Understanding protein function requires knowledge of concentration and interaction with other biomolecules. For decades, scientists have relied on assays to quantify protein levels and study protein behavior.
Bradford Assay for Rapid Protein Quantification
The Bradford assay is one of the simplest and most popular methods for estimating the total protein concentration in a solution. Developed by Marion Bradford in 1976, it exploits the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. In the Bradford reaction, proteins induce a shift in the absorbance maximum and a change in the molar absorptivity of the dye. To perform the assay, diluted Bradford reagent is added to protein samples and standards of a known BSA concentration. After brief incubation, the absorbance at 595 nm is measured. By comparing to a BSA standard curve generated on the same plate, the protein concentration of unknown samples can be determined rapidly in minutes with high sensitivity down to concentrations of 1 μg/ml or less. The Bradford assay is very popular as a simple and inexpensive method for protein quantification prior to experiments. However, it is not very accurate for proteins that differ substantially from BSA in amino acid composition.
Bicinchoninic Acid (BCA) Protein Assay for High Accuracy
The BCA Protein Assays relies on chelation of Cu+1 ions by peptide bonds in proteins under alkaline conditions, followed by a sensitive colorimetric detection of the cuprous cation (Cu+1). In the first step, protein samples are incubated with a solution containing sodium bicarbonate, sodium carbonate, BCA, and a small amount of copper sulfate. Peptide bonds then chelate with Cu+1 ions, reducing Cu+2 to Cu+1. In alkaline conditions, two molecules of BCA can form a complex with one cuprous ion, yielding a soluble purple-colored product that strongly absorbs at 562 nm. By comparing absorbance of samples to a BSA standard curve, protein concentrations can be accurately determined. The BCA assay has advantages over Bradford including greater protein-dye binding stoichiometry, higher sensitivity down to 0.5 μg/ml, and greater linear dynamic range up to 2000 μg/ml. However, it requires about 30-60 minutes to develop full color. Due to its greater accuracy for proteins differing from BSA, the BCA method is preferred for quantitative work requiring a high degree of precision.
Determining Protein Concentration Spectrophotometrically
Many proteins naturally contain amino acid residues like tryptophan, tyrosine and phenylalanine that strongly absorb ultraviolet (UV) light near 280 nm. For such proteins, a simple spectrophotometric method relies on measuring absorbance of diluted protein samples at 280 nm (A280) and using the Beer-Lambert law. The method requires knowledge of the protein's theoretical extinction coefficient based on amino acid composition, which can be calculated using online tools. Beer's law states that A280 = ε x C x l, where ε is the extinction coefficient in M-1cm-1, C is the concentration in mol/L, and l is the pathlength in cm. By rearranging and measuring A280 for diluted samples, protein concentration in mg/ml can be readily determined. Though less accurate than dye-based methods, UV spectrophotometry provides a rapid determination of protein concentration without need for standards. It can be applied to purified proteins but not mixtures due to interference from other compounds absorbing at 280 nm.
Using Protein Gels to Visualize and Estimate Concentration
Gel electrophoresis separates proteins based on size and enables visualization of bands which can then be quantified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) uses SDS detergent to denature proteins and confer a uniform negative charge proportional to molecular mass. Samples are loaded on gels along with precision protein standards of known concentration and molecular weight. After electrophoretic separation, the gel can be stained with Coomassie Brilliant Blue dye to observe protein bands against a blue background. Densitometry of digital gel images allows comparison of band intensities to those of standards and estimation of protein amounts. Though less accurate than assays described above, protein quantification by SDS-PAGE gel staining provides a visual confirmation of purity and approximate concentration along with molecular weight determination. Native PAGE can also be used without denaturation to analyze protein complexes and oligomeric states.
Applications of Protein Assays in Research
Protein quantification is vital at many stages of laboratory research. Bradford, BCA and spectrophotometric assays allow rapid estimation of extracted protein yields from tissues and cells. Accurate protein concentration measurement enables determination and reporting of specific activities for purified enzymes. Protein assays further aid in QC testing for consistency during multi-step purification, characterization of protein-ligand interactions, and assembly of defined reaction conditions requiring precise protein stoichiometry. Measurement of total protein content also serves as an important normalization factor when quantifying and comparing expression levels of target proteins by Western blotting and immunoassays. For proteomics work involving gel-based or mass spectrometry methods, knowledge of protein amounts loaded provides semi-quantitative data for differential expression analysis. Overall, appropriate selection and application of protein assays continues to play a fundamental role across diverse areas of biomedical investigation.
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