HOW TO MEASURE PROTEIN QUALITY

HOW TO MEASURE PROTEIN QUALITY

June 19, 2020 Off By Ava Banks

When working with proteins , the use of impure or low quality samples can lead to the formation of aggregates, precipitates, incorrect folding, loss of activity or even hydrolysis of the same, resulting in inaccurate or low precision experiments.

In this post we review some methods to evaluate the purity of our sample and we will see how to measure the quality of the proteins present in it.

HOW TO MEASURE PROTEIN QUALITY

To assess protein quality, we must examine three fundamental aspects of protein: purity and integrity, homogeneity, and activity.

1.- HOW TO MEASURE THE PURITY AND INTEGRITY OF PROTEINS

Before carrying out any experiments with a protein sample, it is essential to evaluate qualities such as purity and integrity. For this, we can make use of different techniques such as:

  • Electrophoresis

The protein migrates throughout the gel based on its molecular weight, which will allow the presence of contaminants or proteolysis phenomena to be detected, among others.

  • UV-visible spectroscopy

Although this technique is commonly used to measure protein concentration, it is also useful for detecting non-protein contaminants in the sample. This is possible as long as the protein of interest contains aromatic residues and absorbance is monitored over a broad spectrum.

  • Mass spectrometry

Using this method, we analyze the primary structure of the protein, which will allow us to identify whether the protein has undergone phenomena such as proteolysis. Likewise, it allows confirming the presence of post-translational modifications such as glycosylations, phosphorylations, acetylations, etc. in protein.

2.- HOW TO MEASURE THE HOMOGENEITY OF PROTEINS

Once the purity and integrity of our protein sample has been determined, it is necessary to assess whether they are homogeneously distributed in the sample and not, for example, forming aggregates. Let’s see what techniques can be used for this purpose:

  • Dynamic Light Scattering (DLS)

This technique uses polarized laser light to measure the level of diffraction in a sample. The dispersion will be proportional to the hydrodynamic radius of the particles in solution present in the sample, allowing the presence of small amounts of aggregates to be identified.

  • UV-visible and fluorescence spectroscopy

The UV-visible technique allows us to obtain qualitative information about the presence of aggregates in the sample in a simple way, simply by monitoring the absorbance above 320nm. In case of signal, the presence of aggregates is confirmed.

As an alternative, we can also resort to fluorescence spectroscopy . In this case, the aggregation index (AI) is measured, which is the ratio of intensities emitted by the sample at 280nm and 340nm. In the case of an aggregate-free sample, this index must be close to zero.

  • Size exclusion chromatography

It is the standard separation technique to identify and quantify the protein oligomers present in the sample.

3.- HOW TO MEASURE THE ACTIVITY OF PROTEINS

To finish, once the purity, integrity and homogeneity of the protein in the sample have been verified, it is time to evaluate the biological activity and functionality of the same, in the event that this is a requirement for our assay.

The causes that may cause the protein to be non-functional range from incorrect folding during the recombinant expression phase, to interference with additional sequences such as a tag.

To determine the concentration of active or functional protein in the sample, there are multiple specific assays such as cell proliferation assays, enzyme assays, or functional ELISAs , which are generally based on the catalytic properties or binding capacity of the protein of interest to determine the level of biological activity of the same.