PhD student Polly came into the lab on Monday morning to find that her purified protein had started to aggregate after a weekend in the fridge. The profile she got from her Dynamic Light Scattering (DLS) measurement clearly showed two peaks, with the second in the micron range. Her supervisor suggested she tried Nanoparticle Tracking Analysis (NTA) to get more information on the different sized aggregate species in her sample. This showed that there were aggregates of many different sizes present. The question was, what should she do next?
Polly: I’m going to purify another protein batch and then store it in buffer of different recipes. I’ll keep checking that I have my nice DLS monomer peak.
Is there more I can do to understand what’s already happened to my protein?
Prof: So we have a number of other techniques available to us. As well as using DLS and NTA to obtain quick and easy profiles, we can measure relative amounts of monomer, dimer, trimer etc. by SEC (size exclusion chromatography), or use the Archimedes, to investigate larger aggregates by Resonant Mass Measurement (RMM).
This diagram shows how the range of techniques come together to help you see the whole picture:
I suggest that when analyzing your protein, you use a flow chart like this one to decide which technique will give you the information you need (click on the image below to view the full size flow chart):
Polly: What else I could do to characterize the effectiveness of the new storage conditions?
Prof: As well as obtaining size data, we can also measure zeta potential of the protein in each buffer to see if one is potentially more stabilizing than another. Zeta potential can be measured on most Zetasizer instruments and on the NanoSight NS500. Another option is to carry out a thermal stress study, which you can do on these instruments as well. This could predict long term storage stability.
Make sure you read this material, to get a good understanding of each approach to analyzing protein aggregation: