Spectrophotometry and Colorimetric Assays
Measuring the concentration of a protein sample is important because concentration can impact protein aggregation. Ultraviolet-visible (UV-Vis) spectrophotometry – more commonly known as the use of a Nanodrop-like instrument, and colorimetric assays such as Bradford, and BCA assays are the most widely-used methods to measure protein concentration.
In addition, UV-Vis can also be used for the detection of non-protein contaminants (e.g., nucleic acids), and the presence of large particles (e.g., large protein aggregates) by monitoring the absorbance signals at particular wavelengths1.
Electrophoresis is a technique that separates proteins based on their charge and size using electrical input applied to a gel in solution. This can be useful in separating the target protein from non-target proteins which theoretically should bear different physicochemical properties. It is important to have a general idea of the concentration of protein prior to performing electrophoresis as heavily concentrated proteins may precipitate out of solution, aggregates, and/or generally have a difficult time separating in gel. There are many types of electrophoresis techniques, perhaps the most common being polyacrylamide gel electrophoresis.
Dynamic Light Scattering
Dynamic Light Scattering (DLS) is a powerful tool to study diffusion behaviour of proteins based on their size and shape. DLS is often used to probe homogeneity of purified proteins, especially the presence of higher order structures, complexes, and aggregates1. With DSL, the size distribution profile of a protein sample can be determined.
There are many types of chromatography that can be used to separate proteins based on charge, size, and/or their ability to bind a specific protein. Liquid chromatography which includes analytical size exclusion chromatography (SEC) is a routinely used separation technique. SEC, as an example, can be used to detect protein oligomerization and separate different species by their size.
Aggregates and different molecular contaminants present in the protein sample can also be readily separated and quantified with SEC. The most commonly used type of liquid chromatography is High Performance Liquid Chromatography, commonly known by its acronym HPLC. Finally, affinity chromatography can be particularly useful to purify immunoglobulins prior to checking their purity by HPLC, or the aforementioned methods.
Mass spectrometry is an extremely powerful analytical method that can be used to identify proteins based on mass and charge. Protein impurities may be easily detectable with the mass spec depending on the instrument’s resolution, the abundance of the impurity, and the acquisition method of the instrument (e.g., data-dependent acquisition vs data-independent acquisition).