January 1, 1970 (Vol. , No. )

Dave Dilyx Dilyx Biotechnologies

These tips can help you establish conditions that preserve protein function, maintain the integrity of the native fold, and avoid unwanted protein aggregation.

In comparison to DNA samples, protein solutions are much more delicate and therefore require carefully controlled handling procedures. The primary concern is to establish conditions that preserve function, maintain the integrity of the native fold, and avoid unwanted protein aggregation. While the best handling procedures are customized for a specific target protein sample, the following general tips for handling precious protein samples can be applied to all types of proteins.

  1. Safety first. Protein buffers may contain toxic reagents such as the preservative sodium azide or protease inhibitors such as PMSF (phenylmethanesulfonylfluoride)—reagents that are outright toxic. Wearing appropriate protective gear is a must in any protein research laboratory and helps to protect your protein sample from hazards as well.
  2. Storage on wet ice. When conducting experiments, protein solutions are typically stored in containers that rest in a wet water ice bath. Limit handling at room temperature and allow exposure only if stability at elevated temperature has been established.
  3. Long-term sample storage. Shock-freeze large quantities of protein solutions by dropping into liquid nitrogen or by aliquoting 50 microliter amounts into thin-walled PCR tubes that are then dropped into liquid nitrogen. Long term sample storage at -80°C is sufficient to prevent detrimental aging processes such as oxidation or proteolysis.
  4. Prevent sample mix-ups. Every sample has its own history that needs to be captured in your lab notebook. Connect this data with the actual sample at hand by creating two identical labels. One is attached to the tube; the other goes into your lab notebook.
  5. Protect the protein solution from proteolytic breakdown. Reduce any potential protease activity within the protein solution by adding protease inhibitors to the buffer.
  6. Filter out the dust. Remove any dust that may be present in the sample by passing protein solutions through 0.2 micrometer sterile filters. Use containers with tight seals to minimize evaporation and prevent entry of unwanted dust.
  7. Save control samples. Put aside small quantities of protein solutions as well as blank sample buffers as procedures are developed. These samples will come in handy during analytical experiments as negative or positive controls.
  8. Monitor the Protein Aggregation Index. Measuring light absorption of protein solutions at 280 nm and 340 nm is a simple way to track protein sample status. “Healthy” protein solutions have an Aggregation Index of better than 2, whereas values larger than 5 or changes in the Protein Aggregation Index indicate trouble, the latter of which can be fixed by optimizing the protein solubility conditions.

Protein solutions are transparent and clear. Turbidity is a sign of unwanted protein aggregation.

Dave Dilyx ([email protected]) is technical advisor at Dilyx Biotechnologies.

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