The Traditional Method vs. The Filter Vial Method
What is Protein Precipitation?
Protein precipitation is exactly what it sounds like: removing proteins from the solution. This process is often necessary for biological sample analysis, such as in the search for a metabolite of a drug in urine, blood, or serum. The proteins must be removed so that they don't interfere with the analysis and provide a cleaner sample.
How is Protein Precipitation Done?
The most common method of protein precipitation involves adding a precipitant, typically acetyl nitrile, to the sample and vortexing it. This creates a precipitate that can be separated from the supernatant by centrifugation or filtration through a filter vial.
Traditional Method of Protein Precipitation:
- Take a microcentrifuge tube and add 500 μl of acetyl nitrile.
- Add 50 μl of your sample (e.g. urine, blood, serum) to the microcentrifuge tube.
- Vortex the solution for about a minute to precipitate the proteins.
- Centrifuge the solution for about 2-5 minutes at 13,000-14,000 RPM.
- Remove the supernatant (the liquid on top) from the tube and transfer it to an HPLC vial.
- Inject the sample into the HPLC or mass spectrometer for analysis.
Note: This method is not very efficient as the sample may still contain some impurities, but it can still be used for analysis.
Efficient Method of Protein Precipitation using a Filter Vial:
- Take a filter vial and add acetyl nitrile and the sample up to the fill line.
- Vortex the solution to precipitate the proteins.
- Push the plunger of the filter vial partially down, vortex the solution again, and push the plunger completely down to filter the sample.
- The filtered sample is now ready to be injected into the LC or mass spectrometer for analysis.
Note: This method is more efficient as it not only precipitates the proteins but also filters the sample, giving you a cleaner sample for analysis.
Why is Protein Precipitation Important?
Protein precipitation is important because proteins can interfere with the analysis of other compounds present in the sample. This is particularly true in methods such as HPLC or mass spectrometry, where even small amounts of proteins can cause significant interference. By removing the proteins, protein precipitation makes the sample cleaner and ready for analysis.
In conclusion, protein precipitation is an essential step in the analysis of biological samples such as urine, blood, and serum. The process involves removing proteins from a sample to obtain a cleaner solution, making it easier to analyze the desired compounds. There are two common methods of protein precipitation, traditional centrifugation and using a filter vial. Centrifugation involves adding an organic solvent to the sample and vortexing it before spinning it down to separate the proteins and supernatant. On the other hand, the filter vial method involves adding the sample and solvent to a sample cup, vortexing, and then pushing the plunger to filter the sample through a built-in filter, obtaining a cleaner solution. Both methods have their advantages and disadvantages, but the filter vial method is preferred when working with limited sample volume as it provides a cleaner solution with the added benefit of filtration in one step. With the right method and technique, protein precipitation can help scientists obtain accurate and reliable results in their research, contributing to a better understanding of biological processes and potential treatments for diseases.