Deproteinization Using Perchloric Acid Precipitation Protocol

Licia Miller   Product Manager

In biological sample analysis, the presence of proteins often interferes with small molecule detection, and some enzyme activities may affect the analysis results. Perchloric acid (PCA) precipitation has become a widely used method due to its ability to efficiently remove proteins and its stabilizing effect on small molecule analytes (including glycogen, ATP, cAMP, glutathione, antioxidants, etc.).

Perchloric acid precipitation is an efficient and simple protein removal technology. It uses the strong acidity of perchloric acid to destroy the hydrogen bonds and ionic bonds of proteins, denaturing and precipitating proteins, thereby effectively removing most of the proteins in biological samples.

This method can not only quickly enrich small molecule compounds in the sample, such as glycogen, ATP, cAMP, etc., but also stabilize these small molecule analytes during the precipitation process to prevent their degradation. In addition, this method is simple to operate, does not require complex equipment, and the precipitated samples can be directly used for a variety of analytical techniques, such as high performance liquid chromatography (HPLC) and mass spectrometry (MS), which greatly simplifies the sample pretreatment process and improves the analysis efficiency.

Required Materials

- 4 M Perchloric Acid (PCA): Pre-chilled and stored on ice

- 2 M Potassium Hydroxide (KOH): Pre-chilled (for neutralization)

-Microcentrifuge : precooled to 4°C

Phase 1   Protein precipitation

Experimental Steps

1. Sample pretreatment . Take about 1 mL of protein-containing sample, ensure that the protein concentration in the sample is at least 5 mg/mL, homogenize and centrifuge the sample to ensure a clear protein solution. The sample must be kept on ice throughout the process.

2. Add PCA. Add PCA to the homogenate to a final concentration of 1 M and vortex briefly to mix.

Note: Samples with high protein concentrations may require an increase in the amount of PCA.

3. Incubation and centrifugation . Incubate on ice for 5 minutes, then centrifuge at 13,000 × g for 2 minutes at 4°C. Transfer the supernatant to a new tube.

Phase 2   Sample Neutralization

Experimental Steps

1. Add pre-cooled 2 M KOH at 34% of the supernatant volume (e.g., add 34 μL KOH to 100 μL sample) and vortex briefly to mix.

Note: Gas (such as CO₂) may be produced during the neutralization process and needs to be vented.

2. Use pH test paper to test 1 μL of sample to ensure the pH is between 6.5 and 8.0. If necessary, use 0.1 M KOH or PCA to make fine adjustments.

Centrifuge at 13,000 × g for 15 minutes at 3.4°C and collect the supernatant. At this point, the sample has been deproteinized, neutralized, and PCA removed and can be used directly for subsequent testing.

Phase 3   Sample Recovery

After protein removal, the sample will be diluted and the original concentration should be calculated using the following formula:

Original concentration percentage = (initial sample volume / (initial sample volume + PCA volume + KOH volume)) × 100

Advantages and Scope of Application

1. Efficient protein removal: PCA can remove most of the proteins in the sample and is suitable for quantitative pretreatment of various small molecules such as glycogen, ATP, cAMP, and glutathione.

2. Strong stability: PCA can not only precipitate proteins, but also stabilize small molecule analytes and reduce the risk of degradation.

3. Operational standardization: Combine centrifugation and neutralization steps to ensure experimental repeatability and suitable for high-throughput detection.

Precautions

Avoid interfering substances: If the sample lysis buffer contains EDTA or sodium vanadate, it may inhibit PCA activity and the reagent formula needs to be adjusted.

pH control is key: pH after neutralization needs to be strictly tested to avoid affecting subsequent tests (such as antibody binding or enzyme activity).

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/