We measure protein using various methods, and the appropriate method depends on the nature of the protein and the context of the measurement. For purified proteins, UV absorbance is often the method of choice.
Measuring Protein: Key Methods
Here's a breakdown of how we measure protein, focusing on UV absorbance as a popular method:
UV Absorbance
- Principle: Proteins absorb UV light, particularly at a wavelength of 280 nm due to the presence of aromatic amino acids (tryptophan, tyrosine, and phenylalanine). By measuring the absorbance at 280 nm, we can quantify the protein concentration.
- Advantages:
- Fast and convenient.
- Reproducible results.
- Non-destructive; it does not consume the protein sample.
- Requires no additional reagents, standards, or incubation periods.
- Applicable for purified proteins.
- Example Application: A researcher wants to determine the concentration of a purified protein after chromatography. UV absorbance is an ideal method because the protein is already isolated, the measurement is fast and the protein sample will not be lost.
- Considerations:
- Absorbance at 280nm is dependent on amino acid composition (aromatic amino acids contribute most to absorbance at 280nm), and so the extinction coefficient, specific for each protein, should be used for precise measurements.
- Other molecules can also absorb at 280 nm (such as nucleic acids), which can interfere with measurements if the sample is impure.
Other common methods for Measuring Protein
Besides UV absorbance, other methods for protein measurement are also used. Here are a few:
- Colorimetric Assays (e.g., Bradford, Lowry, BCA): These methods involve chemical reactions that produce a colored product, the intensity of which is measured using spectrophotometry. They are good for both purified and unpurified protein samples.
- Bradford assay: uses the binding of a dye to proteins to measure the absorbance at 595 nm.
- Lowry assay: uses a more complex colorimetric reaction and measures absorbance at 750 nm.
- BCA assay: is very similar to Lowry but more accurate, and measures absorbance at 562 nm.
- Mass Spectrometry: Highly sensitive technique for measuring protein abundance and modifications, useful for complex mixtures.
- Immunological methods: ELISA, Western blot, and other techniques that use antibodies specific for the protein of interest, allowing for detection, identification, and quantification.
| Method | Principle | Advantages | Disadvantages | Sample Type |
|---|---|---|---|---|
| UV Absorbance (280nm) | Protein absorption of UV light at 280nm. | Fast, convenient, reproducible, non-destructive, no additional reagents. | Can be affected by sample purity. Depends on amino acid composition. | Purified proteins |
| Bradford Assay | Binding of dye to protein causing a change in absorbance. | Simple, fast, relatively inexpensive. | May be affected by detergents and some other chemicals, less sensitive. | Purified and unpurified samples |
| Lowry Assay | Chemical reaction leading to a colored product. | More sensitive than Bradford. | More time-consuming, susceptible to interference, not compatible with all chemicals. | Purified and unpurified samples |
| BCA Assay | Chemical reaction similar to Lowry, with better stability. | More accurate and stable than Lowry. | Susceptible to interference. | Purified and unpurified samples |
| Mass Spectrometry | Measure mass-to-charge ratio of protein fragments. | Highly sensitive, can identify and quantify multiple proteins. | Complex process, can be expensive. | Complex samples, purified proteins |
| Immunological Methods | Antibody recognition and binding to the protein of interest. | Highly specific and can be used in complex samples. | Requires the availability of antibodies, may require multiple steps. | Complex samples, purified proteins |
Summary of how to measure protein
For purified proteins, UV absorbance is a popular method due to its convenience, speed, and non-destructive nature. Colorimetric assays such as Bradford, Lowry, and BCA are widely used for other types of samples. Mass spectrometry and immunological methods offer more sophisticated means for measuring protein concentration.
