A growing field of research is dedicated to protein aggregation, which can be useful, functional or unwanted. This article explores some of the reasons why proteins aggregate, and details the techniques available to study it.
The good, the bad and the ugly
Protein aggregation is often seen as an unwanted interaction between protein monomers. Yet a growing field of research is dedicated to understanding the mechanisms of this phenomenon, which can be useful, functional or unwanted.
In some instances, aggregation can present benefits; proteins that aggregate are utilized in the food industry where they are desired for their texture and flavor. By contrast in the pharmaceutical industry, where large scale production of protein-based drugs is the goal, protein aggregation is a major problem. It can go either way in biology, where aggregates may be functional, or for example amyloid-ß, play a role in disease.
So whether you are studying the good or bad, or just happen to have an unintended ugly aggregate, understanding and being able to detect aggregation is a vital part of many scientific studies.
What causes protein aggregation?
Some of the most common reasons proteins aggregate are;
- High concentration, a common problem found when overexpressing protein in bacteria.
Conformational change, a switch from native to denatured that results in hydrophobic patches being exposed and aggregates being formed.
- Chemical changes, such as oxidation, or physical changes, such as interacting with surfaces.
However, there are many factors that can contribute to protein aggregation including ionic strength, pH, temperature, agitation, freeze-thaw cycles and even the container you store your protein in. Any or all of these can be exploited to induce or inhibit aggregation.
How to study protein aggregation
There is no shortage of techniques for studying protein aggregation, whether you are detecting the presence of aggregates, or following the aggregation process. Often, the first time aggregates are detected are when they are visible in solution to the naked eye, either as precipitates or just a cloudy solution, but there are a host of other techniques that are more reliable and sensitive.
Visual and microscopic techniques
While some aggregates can be seen with the naked eye, microscopic inspection is more often utilized to detect smaller aggregates. Aggregates greater than 1 µM can be detected using a standard light microscope. Greater detail can be achieved by utilizing more sensitive microscopes, with a variety of fluorescent techniques available that are capable of detecting the interaction between two molecules, while both electron microscopy and atomic force microscopy are capable of providing fine detail and structural information about your aggregates.
- If your sample if cloudy or has visible aggregates, then a simple visual inspection is cheap and very quick.
- Most labs have access to standard light/fluorescent microscope.
- Detailed structural information if using electron or atomic force microscopy.
- Typically one sample analyzed at a time
- Fluorescent molecules may alter aggregation properties of protein
- More specialized microscopy techniques can require high technical expertise
Molecular probes such as ThT, congo red and ANS can be used to monitor the aggregation process in real time. The exact mechanism of action for each of these dyes is debated and in some concentrations, these dyes have even been suggested to reduce aggregation. Nonetheless, they are widely used in aggregation studies, and with continuous measurements allow for detailed kinetic studies on aggregation.
- Aggregation pathway followed in real time
- Multi-well format allows for several conditions to be measured at the same time
- Presence of additives (e.g. lipid vesicles) unlikely to interfere with measurements
- No structural information
- Dyes used may influence aggregation
- Early aggregation events not detectable
Techniques based on separation include electrophoresis, both SDS-PAGE and Native-PAGE, size exclusion chromatography and ultracentrifugation. These techniques rely on the size and conformation of a protein changing how it moves through a defined medium.
- Gives information on size of aggregates and potentially on intermediate species
- SDS-PAGE very common and easy technique
- Electrophoresis can be high throughput depending on set-up with commonly used set-ups capable of running 4 gels of 10-20 lanes each at a time
- Low throughput, one sample at a time for size exclusion chromatography
- Centrifugation limited by rotor type
- Time consuming
- End point measurements – often by the time the test is complete, it is only representative of the end point
The ability of particles to block or scatter light can also be utilized by techniques ranging from dynamic light scattering (DLS) & nanotracking analysis (NTA) to small angle scattering with X-rays (SAXS) or neutrons (SANS). While DLS can be adapted to high-throughput, multi-well plate readings, other scattering techniques are typically used to analyze one sample at a time.
- Small aggregates detected
- Aggregation can be followed over time
- Structural information or size provided
- Large aggregates can swamp signal
- Sample needs to be highly pure
- Can require large amounts of protein
- Can be technically challenging
There are a host of other techniques that can be utilized for studying protein aggregation, including circular dichroism (CD), NMR, mass spectrometry and raman spectroscopy. Since these require more specialized equipment and training we will not discuss them in detail here.
Microfluidic Diffusional Sizing (MDS)
Microfluidic Diffusional Sizing (MDS), such as on the Fluidity One, allows you to perform a quick analysis on your protein of interest to check for aggregation, and the simplicity and speed means you can perform repeated measurements over time to follow the aggregation process.
A big advantage of Fluidity One is the small amount of material needed. If we compare Fluidity One to ThT assays, 4 µg of α-synuclein is needed per well in a typical ThT assay, compared to 50 ng of protein per chip for MDS. With no additional preparation needed, just pipette your protein of interest directly onto the chip, MDS makes an ideal all-in-one technique to begin your aggregation studies.
The need to study protein aggregation
Whichever technique you chose to use to study protein aggregation comes down to what information you want and what equipment and expertise you have available.
Maybe you just want to check the quality of your protein preparation, in which case you want a quick and potentially high throughput technique; essentially, you want to know “do I have protein aggregates?”. MDS allows you to answer this question quickly and easily.
If you want more information about your aggregates, or to understand how your protein aggregates, then more in-depth or multiple orthogonal techniques are likely to be employed, addressing both the structure and kinetics of your aggregates.
Get in touch here to ask our applications scientists anything about protein aggregation analysis.