Detect, quantitate and analyze proteins
Protein detection, quantification, and analysis are central to investigating a wide variety of biological processes. Measuring the protein concentration is necessary to processes ranging from protein purification and labeling to sample preparation for electrophoresis.
Protein quantification is the measurement of the total protein concentration in a sample. Protein can be quantitated directly via absorbance at 280 nm or indirectly using colorimetric (BCA, Bradford, etc.), or fluorometric methods offering advantages, such as greater sensitivity. To identify and measure a specific protein within a complex sample, for example, serum or cell lysate, an enzyme-linked immunosorbent assay (ELISA) may be used.
Cellular signaling and other biological processes may be analyzed using fluorescent proteins. For example, green fluorescent protein (GFP) can be expressed in living cells and used to visualize the localization and protein dynamics under experimental conditions. Tryptophan, an amino acid whose fluorescence emission properties are affected by its microenvironment, has also been used to gain information on changes in the conformational state of proteins.
Protein quantification and analysis methods
Microplate readers with absorbance, fluorescence, and luminescence detection modes provide a versatile solution for the quantification and study of proteins. Our application notes demonstrate how these instruments can be used to obtain and analyze results.
FAQs
Determining the concentration of protein samples generally is accomplished either by measuring the UV absorbance at 280 nm or by reacting the protein quantitatively with dyes and/or metal ions (Bradford, Lowry, or BCA assays).
What is the most accurate protein quantification? ›
Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.
What is the best way to quantify protein expression? ›
Protein quantification techniques can include bicinchoninic acid assay (BCA), variations of high-performance liquid-based chromatography (HPLC) and the use of fluorescently labelled or radio-chemically labelled proteins.
What does protein quantification tell us? ›
Protein quantification is the measurement of the total protein concentration in a sample. Protein can be quantitated directly via absorbance at 280 nm or indirectly using colorimetric (BCA, Bradford, etc.), or fluorometric methods offering advantages, such as greater sensitivity.
What are the advantages of BCA assay? ›
Advantages of BCA assays
One of the major advantages of the BCA assay is that it produces a linear response curve. This response curve allows accurate determination of unknown protein concentrations and provides a higher dynamic range than other standard assays.
What does SDS PAGE tell you? ›
Page Contents. SDS-PAGE is a reliable method for determining the molecular weight (MW) of an unknown protein, since the migration rate of a protein coated with SDS is inversely proportional to the logarithm of its MW.
Why is it important to accurately quantify proteins? ›
It enables users to compare results between experiments or between proteins with greater precision. It can be used to determine protein yield, measure enzyme kinetics, and during protein extraction it can confirm the success of cell lysis.
How long does protein quantification take? ›
First, the Bradford protein assay is much easier to use. It requires one reagent and 5 minutes to perform as compared to the three reagents and 30–40 minutes typical for the Lowry assay.
What is the most commonly used tool to measure protein quality? ›
6.2.1.4.
The Kjeldahl method is widely used internationally and is still the standard method for comparison against all other methods. Its universality, high precision and good reproducibility have made it the major method for the estimation of protein in foods.
What methods are used to quantify protein? ›
The most frequently used methods for measuring protein content in foods include the Kjeldahl method, Dumas method, direct measurement methods using UV-spectroscopy and refractive index measurement.
There are two methods that are commonly used to identify proteins: Edman Degradation and Mass Spectrometry.
What are four ways to quantify the amount of protein in a sample? ›
Top 5 Protein Quantification Assays
- UV-Vis Absorbance at 280 nm. ...
- The Bradford Assay. ...
- The Bicinchoninic Acid (BCA) Assay. ...
- The Folin–Lowry Assay. ...
- The Kjeldahl Method.
Western blot is an invaluable lab technique used to detect proteins in a tissue or blood sample. It helps researchers identify specific protein molecules in a complex mixture of proteins. Since antibodies are used in this technique to mark the target protein, this technique is also known as an immunoblot.
How can proteins be identified? ›
Mass spectrometry (MS) is a commonly used, high-throughput tool for studying proteins. The procedure of MS-based protein identification involves digesting proteins into peptides, which are then separated, fragmented, ionised, and captured by mass spectrometers.
