Deproteinization

This deproteinization protocol provides a reliable method for removing proteins from biological samples using perchloric acid (PCA) precipitation. Total protein extraction is a critical precursor step for many sample types, and this protocol is designed to follow or complement such extraction processes. This technique is essential for preparing samples for small molecule analysis, ensuring accurate and interference-free results. The protocol includes three main stages: protein precipitation, sample neutralization, and recovery. It is compatible with various sample types, including serum, tissue homogenates, and cell lysates. Designed for ease of use and reproducibility, the protocol supports downstream applications such as ATP, cAMP, and antioxidant quantification.

Introduction

Most studies report protein interference as a common challenge in biochemical assays, especially when analyzing small molecules. Various extraction methods are available for sample preparation, but PCA precipitation is particularly effective for protein removal. This deproteinization protocol addresses this issue by employing PCA precipitation, a well-established method for protein removal. It is particularly useful for researchers conducting metabolite quantification or enzymatic assays. By following a simple and scalable procedure, users can achieve consistent deproteinization across a wide range of biological samples. The protocol is optimized for both manual and kit-based workflows, making it suitable for routine lab use.

Background and principles

Deproteinization is a critical step in sample preparation for biochemical assays. Protein extraction is often the initial step in preparing samples for analysis, and contaminants such as DNA and salts can interfere with downstream applications. This protocol utilizes PCA to precipitate proteins, which are then separated by centrifugation. PCA not only removes proteins but also stabilizes small molecule analytes, preserving their integrity for downstream analysis. PCA precipitation is effective at removing not only proteins but also nucleic acids, especially DNA, which can interfere with protein separation and analysis. After precipitation, the sample is neutralized using potassium hydroxide (KOH), ensuring compatibility with assay conditions. The removal of salts and chaotropic agents is also important for improving assay resolution and reproducibility. This method is favored for its efficiency, simplicity, and ability to maintain analyte stability. Compared to filtration or enzymatic digestion, PCA precipitation offers a faster and more reproducible approach to deproteinization.

Materials

• 4 M perchloric acid (PCA), ice cold
• 2 M KOH, ice cold (neutralizing agent)
• Microcentrifuge (set at 4°C and previously cooled down)
• Ice
• Microcentrifuge tubes​

Stage 1 - Protein precipitation

Steps

Prepare your sample as specified in the product protocol.

• You should have a clear protein sample after homogenization and centrifugation.
• Keep your samples on ice.

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

Incubate samples on ice for 5 min.

• Centrifuge samples at 13,000 x g for 2 min in a cold centrifuge.
• Transfer the supernatant to a fresh tube.

Stage 2 - Sample neutralization

Steps

Neutralize supernatant by adding ice cold 2 M KOH that equals 34% of the volume of the supernatant to your sample (for example, add 34 µL of 2 M KOH to 100 µL of sample) and briefly vortex.

• This will neutralize the sample and precipitate excess PCA.
• There may be some gas (CO₂) evolution so vent the sample tube.

After neutralization it is very important that the pH equals 6.5 - 8.0 (use pH paper to test 1 µL of sample).

• If necessary, adjust the pH with 0.1 M KOH or PCA.

Spin samples at 13,000 x gfor 15 min in a cold centrifuge.

• Collect supernatant.

Stage 3 - ​Sample recovery

The deproteinized samples will be diluted from the original concentration. To calculate the dilution factor of your final sample, simply apply the following formula:

% original concentration = (initial sample volume / initial sample volume + volume PCA + volume KOH) x 100

Sample recovery

The sample recovery stage is a pivotal part of the sample preparation process, especially when preparing samples for combined chemical analysis and in vitro bioassays. After the initial extraction and deproteinization steps, it is essential to recover the sample in a form that is both clean and concentrated, ensuring that biologically relevant compounds and spiked genotoxic compounds are retained for accurate analysis. Techniques such as centrifugation, filtration, and evaporation are commonly used to remove excess solvents and concentrate the sample, resulting in a solution that is suitable for both chemical analysis and bioassay testing.

Comparison to other methods

The PCA-based deproteinization protocol stands out for its dual function: protein removal and analyte stabilization. Compared to trichloroacetic acid (TCA) precipitation, PCA is less harsh and better suited for preserving sensitive molecules. When compared to solid phase extraction, PCA precipitation offers a simpler workflow and is less equipment-intensive, though solid phase extraction provides higher recovery rates and broader chemical compatibility, especially for extracting a wide range of analytes from environmental water samples. Filtration methods, such as using a 10 kDa cutoff membrane, can remove up to 98% of proteins but are more time-consuming and equipment-dependent. Enzymatic digestion, while effective, may introduce variability and require additional purification steps. Alternative extraction methods often utilize an organic solvent, such as DMSO, which must be carefully selected to ensure compatibility with downstream chemical analysis and in vitro bioassays. The performance of each method, including PCA precipitation, solid phase extraction, and organic solvent-based extraction, can be evaluated using spiked compounds to assess recovery and detection efficiency. PCA precipitation achieves approximately 95% protein removal with minimal sample loss and is compatible with a wide range of assays, making it a preferred choice for many researchers.

Applications

Deproteinization is widely used in the preparation of samples for small molecule quantification. It is suitable for assays measuring ATP, cAMP, glutathione, glycogen, and antioxidants. Researchers working with serum, urine, tissue homogenates, or cell lysates can benefit from its versatility.

The protocol is highly compatible with in vitro bioassay applications, supporting a wide range of bioassays such as CALUX assays, including ER CALUX, AR CALUX, GR CALUX, and PR CALUX assays, for detecting specific endocrine receptor activation and agonistic activities in environmental and biological samples. It is also suitable for genotoxicity assays, including the comet assay and Ames fluctuation test, to detect DNA damage and mutagenic potential. This protocol enables researchers to determine biologically relevant compounds in surface water and other sample types, with samples able to be chemically analyzed and compared to predicted responses from bioassays. Additionally, it supports the detection of antagonistic effects in bioassays and can be used as part of a powerful toolbox for comprehensive water quality assessment.

The protocol is also applicable in cancer research, metabolic studies, and pharmacological screening. Its compatibility with various assay formats, including colorimetric and fluorometric kits, makes it a valuable tool in both academic and industrial laboratories. Ensuring clean and stable samples enhances the reliability of analytical results.

Limitations

While effective, the PCA deproteinization protocol has some limitations. It requires careful handling of corrosive reagents like perchloric acid and potassium hydroxide. The protocol may not be suitable for analytes sensitive to acidic or basic conditions. Sample dilution during the process can affect assay sensitivity, requiring correction factors. Additionally, precise pH adjustment is critical to avoid assay interference. Users must ensure proper centrifugation and temperature control to maintain reproducibility. Despite these considerations, the protocol remains a robust option for most small molecule analyses when used with appropriate precautions.

Troubleshooting

Common issues with the PCA deproteinization protocol include incomplete protein precipitation, incorrect pH adjustment, and sample loss during centrifugation. If protein remains in the supernatant, increase PCA concentration or extend incubation time. For pH deviations, verify the accuracy of KOH addition and use pH paper to confirm. Excess gas formation during neutralization can be mitigated by carefully venting the tubes. If sample recovery is low, check centrifuge settings and ensure tubes are pre-cooled. Dilution errors can be corrected using the provided formula. Abcam’s technical support and detailed kit instructions offer additional guidance for resolving protocol challenges.

The Abcam deproteinizing sample preparation kit is not intended for industrial or diagnostic use, and is only recommended for research purposes.