ReadiUse™ TCA Deproteinization Sample Preparation Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
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H-phrase	H303, H313, H340
Hazard symbol	T
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R68
UNSPSC	41116134

Overview SDS Protocol

The analysis of amino acids, ions, metabolites and other small molecules is frequently hindered by the interference of lipids, protein and enzymes present in biofluids, cell and tissue lysates. Therefore, deproteinization is a necessary step in many procedures prior to analysis of biological samples. AAT Bioquest's Deproteinization Sample Preparation Kit offers a rapid method for removing proteins in biological samples. Proteins are precipitated by applying trichloroacetic acid (TCA). After removal of precipitated proteins, the pH of the sample is neutralized with neutralization solution. TCA based deproteinization has been successfully and widely used in sample preparation prior to quantitation of small molecules, such as glycogen, ATP, cAMP, glutathione and antioxidants. The TCA method provided in this kit can also be used for removing protein in large scale. Biological samples prepared using this kit can be directly used in various biochemical analysis.

Components

Example protocol

AT A GLANCE

Important notes
Place kit components on ice before use.

SAMPLE EXPERIMENTAL PROTOCOL

Protein Precipitation:

Prepare a clear solution sample with protein concentration less than 50 mg/mL after homogenization and centrifugation. Keep protein samples on ice. Note: High protein concentration samples might need to be diluted.
Add 10 µL of cold TCA (Component A) into 100 µL of protein sample and vortex briefly to mix well.
Keep the sample on ice for 10 minutes. Centrifuge at 12,000 rpm for 5 minutes. Transfer supernatant (deproteinized sample) to another tube. Note: It is recommended to neutralize samples in TCA and use for relevant assays immediately. However, deproteinized samples in TCA may be stored at -70°C for up to one month.

TCA Neutralization:

To neutralize excess TCA, add 10 µL of cold Neutralization Solution (Component B) into the collected supernatant and vortex briefly to mix well.
Keep the samples on ice for 5 minutes. Samples are now deproteinized and neutralized.
(Optional) Read absorbance at 280 nm (OD) to check if the samples are deproteinized.

Images

Figure 1. TCA-based deproteinization of protein samples. Bovine serum albumin (BSA) samples with protein concentration less than 50 mg/mL and a rabbit serum sample at the concentration about 14.2 mg/mL were deproteinized using ReadiUse™ TCA Deproteinization Test Sample Preparation Kit (Cat#19501). More than 98% of protein in all samples was removed with TCA method.

Citations

View all 2 citations: Citation Explorer

Curcumin induces apoptosis by inhibiting BCAT1 expression and mTOR signaling in cytarabine-resistant myeloid leukemia cells
Authors: Tseng, Yu-Hsin and Yang, Rei-Cheng and Chiou, Shyh-Shin and Shieh, Tzong-Ming and Shih, Yin-Hwa and Lin, Pei-Chin
Journal: Molecular Medicine Reports (2021): 1--8

Analysis of the Status of the Cutaneous Endogenous and Exogenous Antioxidative System of Smokers and the Short-Term Effect of Defined Smoking Thereon
Authors: Lohan, Silke B and B{\"u}hring, Karl and Lauer, Anna-Christina and Friedrich, Annette and Lademann, J{\"u}rgen and Buss, Annette and Sabat, Robert and Wolk, Kerstin and Meinke, Martina C
Journal: Antioxidants (2020): 537

References

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