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Five Methods for Assessing Protein Purity and Quality

Posted in: Protein Expression and Analysis

Content sponsored by Fluidic Analytics

3 test tubes, one with GFP fluorescing

If you’ve ever worked with proteins in the lab, you probably know just how critical protein purity and quality are for downstream applications. In this article, we’ll review the multitude of problems that are encountered with ‘bad’ protein samples and how you can analyze the purity and integrity of your favorite protein prior to using it for important experiments.

Issues Resulting from Poor Protein Sample Quality

Whether your protein is impure or of poor quality, bad samples result in a plethora of issues that lead to inaccuracy and low precision across experiments—and can sometimes cause experiments to fail completely! Here are a few specific issues to watch out for:

  1. Proteins can misfold, aggregate, and precipitate out of solution, which is often an irreversible process when not carefully controlled (such as in a salt precipitation). Aggregation and degradation also causes proteins to not ‘behave’ as they would in their native state, so they may not bind to other proteins or molecules correctly. Sometimes aggregation is clearly visible in a sample by eye, but this is not always the case.
  2. When enzymes become inactivated or misfolded, enzymatic assays fail or result in poor reproducibility. This can be of particular importance if you are using enzymatic assays as indicators for other proteins of interest!
  3. Hydrolysis in proteins leads to the above issues in addition to risking the loss of carefully constructed functional tags. While there are agents that reduce hydrolysis and degradation in protein samples, there is always the risk of hydrolysis whenever your protein is stored in an aqueous solution.

Clearly, it’s important to invest some time into carefully controlling the factors that influence protein quality. You likely have some control over storage conditions, including pH, buffer, salt concentration, protein concentration, and storage temperature. With that said, protein quality can change over time even with the most optimal preparation and storage conditions. This means that you should check your protein samples regularly to ensure they are up-to-scratch for your experiments!

Taking a moment to check the quality of your protein before embarking on time consuming or costly techniques can really help to save both time and money in the long run.

Reliable Methods for Measuring Protein Purity and Quality

  1. General Quantification: UV-Vis, Bradford, and Activity Assays

Measuring the concentration of a protein sample is generally required for many of the methods outlined later in this article to be effective.

While UV-Vis spectrophotometry and Bradford assays are high-throughput and are used in almost every biochemistry lab, they are relatively crude compared to enzymatic activity assays. This is because UV-Vis and Bradford assays results depend on the total protein within a sample, not just your protein of interest. In contrast, activity assays are target specific and have the additional benefit of measuring the fraction of active protein in a purified sample. However, not all proteins can be quantified with an activity assay.

  1. Size Analysis: Electrophoresis (Native/Denaturing PAGE)

Like the quantification methods described above, electrophoresis is widely employed by biochemists and can provide a general picture of both the size of your target protein and whether there are other protein-based impurities present. However, you’ll want to get an approximation of how concentrated your protein is before performing electrophoresis—lest you risk having to repeat running a gel at 6:30pm on a Friday night!

There are several types of electrophoresis methods, the most common being denaturing SDS-PAGE. Samples are first denatured with SDS (a detergent) then separated by mass on a polyacrylamide gel matrix using an electric field. In native PAGE, protein separation is more complex and is based on net charge, size, and shape of the native structure. In both techniques, you can identify if degradation is present in a sample by a ‘smeared’ band- but this can also occur if you have overloaded the gel with too much protein.

Some challenges of electrophoresis include that it does not reveal low-level impurities or minute size differences. Electrophoresis also requires samples with a concentration between 0.1 and 2 mg/mL to provide clear results.

  1. (More) Size Analysis: Mass Spectrometry

Mass spectrometry is a very powerful analytical technique that can identify post-translational modifications with great accuracy and precision, which are not easily visualized with the techniques described above. Ionizing mass spectrometry works by separating proteins or peptides by mass and charge, accelerating them onto a detector, and creating a unique spectrum for each protein (or protein fragment).

The drawbacks of relying solely on mass spectrometry for assessing protein quality is that it is relatively low-throughput and requires extensive sample preparation. In addition, it is difficult to assess whether proteins in a sample are intact, since the process is denaturing and does not identify misfolding events.

  1. Homogeneity: Dynamic Light Scattering

If your protein appears pure using the methods above, it’s time to check to ensure that it is dispersed within the sample (i.e., not aggregating). Dynamic light scattering (DLS) uses polarized laser light to measure the level of diffraction in a sample with small molecules (or in our case, proteins!). The amount of scattering that occurs is an effect of the hydrodynamic radius of the particles in solution as the sample travels through the instrument.

While DLS is an easy-to-use technique that provides excellent qualitative information, it doesn’t provide a totally comprehensive picture of the size distribution in a protein sample since aggregates can easily overwhelm the detector. This technique is also not suitable for assessing quaternary structures (i.e., dimers versus monomers). With that said, the convenience of DLS combined with its ability to reveal aggregate formation over time makes it a widely used method for assessing homogeneity.

  1. Microfluidic Diffusional Sizing (MDS) (Fluidic Analytics)

Microfluidic diffusional sizing (MDS) as used in the Fluidity One system by Fluidic Analytics is a fast and simple option to measure protein size and concentration, which together give a good indicator of quality. MDS uses microfluidic chips to run the protein sample into a channel where it flows alongside an auxiliary fluid in a steady state laminar flow – with no mixing. The only way proteins can move from one stream to the other is by diffusion, which occurs at a rate proportional to their size (hydrodynamic radius, Rh) After some diffusion, the two streams are split again and the proteins labelled. The ratio of diffused and undiffused is used to calculate the Rh.

MDS avoids some pitfalls of other technologies. There is no interaction between the protein and a matrix, like in electrophoresis, and samples are run in their native state. The workflow required is simple with results under 10 minutes using samples with concentrations as low as ~10µg/mL.

Which Method Should I Use to Analyze My Protein?

It can be overwhelming to choose techniques to measure the quantity, purity, and overall quality of a protein sample- but doing so will save you a lot of headaches down the line! Though there are many techniques available, you don’t have to choose just one— all of the techniques described above can be used in tandem with one another to provide the most accurate and comprehensive picture of the state of protein samples.


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Image Credit: Ryan Kitko

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