Protein precipitation

Stephanie J. Marin, Ph.D.

Protein precipitation vs. traditional protein crash: what's best?

October 7, 2020 at 8:00 AM / by Stephanie J. Marin, Ph.D.

Most clinical chemists have developed a blood, serum or plasma assay using a protein crash because it is inexpensive and generally removes proteins that interfere with detection or the analysis in some way. But is this always true?

Protein crash

It's usually done by adding a crash solvent like methanol or acetonitrile to the blood sample, vortex mixing, then centrifuging and removing the supernatant to remove the crashed proteins from the sample.

Sometimes the crash solvent is acidified to aid solubility and begin the denaturation process. The crash solvent is usually added at a ratio of 3:1, but can sometimes require up to 10:1 solvent:specimen.

For more information on the process of doing a protein crash I recommend this article:

Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis

Protein precipitation

It's a simple, yet effective sample preparation treatment. It provides very minimal clean up, but can be sufficient for some clinical applications. In my work, I developed and validated a protein precipitation method for a serum/plasma drug panel, and it worked great ... for awhile.

All of the steps were manually done. The addition of specimen and solvent could have been done using a liquid handler, but at some point the samples had to be vortexed or shaken, put in a centrifuge, removed and the supernatant poured off. The process was time-consuming and we had issues with LC column plugging. We used guard columns to protect the LC column, but we were changing those frequently. This is a scenario that often results from samples that aren't sufficiently cleaned up.

I have since learned that sample throughput and sample cleanliness can be improved with the use of a protein precipitation plate, like the ISOLUTE PPT+.  The ISOLUTE PPT+ contains a course porosity frit designed to trap crashed proteins, combined with a functionalized frit designed to hold up a volume of crash solvent until vacuum or positive pressure is applied. Crash solvent is added to the wells of the PPT+ plate, then specimen is added to the solvent and protein precipitation occurs. Vacuum or positive pressure is applied to force the clean extract through the frits, filtering the crashed protein out of the sample.

This solvent first methodology is designed to create maximum protein crashing without the need for offline vortexing. 

  • Acetonitrile is typically added at a volume of 3:1 solvent:specimen
  • Methanol is used at a ratio of 4:1 to 10:1
  • TCA or perchloric acid is effective for whole blood specimens

A protein precipitation plate can produce a cleaner sample than a manual protein crash and allows automation in a 96-well format. It eliminates cloudy extracts, manual shaking or vortexing, centrifuging and supernatant transfer.  For high-volume, high throughput assays, it produces a cleaner sample, with a more efficient workflow compared to a manual protein crash.

To know more about protein precipitation and other sample preparation techniques, click on the button below.

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Topics: Sample preparation, Method development, Protein precipitation