Deproteinization

Last edited Tue 16 Mar 2021

The analysis of small molecules in biological samples is frequently hindered by the presence of protein and various enzyme activities. Many bioassays require removal of protein from samples prior to analysis. Perchloric acid (PCA) precipitation is one of the most extensively used deproteinization protocols since it not only removes most of the protein present in the sample but it also functions to stabilize many of the small molecule analytes.

PCA deproteinization has been successfully used in the preparation of samples prior to quantitation of an array of small molecules, including glycogen, ATP, cAMP, glutathione, antioxidants, etc.

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 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 supernatant to your sample (for example, add 34 µL of 2M 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 g for 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