May 17, 2023
Simple TCA/acetone protein extraction protocol for proteomics studies.
- Angelo José Rinaldi1
- 1Universidade Federal de Viçosa
Protocol Citation: Angelo José Rinaldi 2023. Simple TCA/acetone protein extraction protocol for proteomics studies.. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8j74dg2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 29, 2023
Last Modified: May 17, 2023
Protocol Integer ID: 81176
Keywords: acetone protein extraction protocol for proteomics study, acetone for protein extraction, acetone protein extraction, acetone protein extraction protocol, protein extraction with tca, protein extraction, effective in selective protein precipitation, selective protein precipitation, important in proteomic study, proteins from biological sample, high quality proteins for various analyze, proteomics study, proteomic study, analysis of protein, obtaining protein, obtaining high quality protein, protein purity, extraction, preservation of protein structure, processing biological sample, extraction protocol, biochemical analysis, proteins of interest, protein, use of tca, removal of contaminant, removal of lipid, removing lipid, acetone, removing contaminant, biological sample, preserving protein structure, protein structure, trichloroacetic acid
Abstract
Protein extraction with TCA (trichloroacetic acid) and acetone is a widely used method to precipitate proteins from biological samples. This method is useful for obtaining proteins of interest for proteomic studies or biochemical analyses. Here are some reasons why TCA/acetone protein extraction is important:
Selective Protein Precipitation: TCA/Acetone is effective in selective protein precipitation, allowing the removal of contaminants and unwanted components present in the sample. This is especially important in proteomic studies, where protein purity is critical for subsequent analyses.
Removal of lipids and carbohydrates: TCA/acetone is efficient in removing lipids and carbohydrates, which can interfere with proteomic and biochemical analyses. These compounds are precipitated together with the proteins, facilitating their removal and improving the quality of the samples.
Preservation of protein structure and function: The use of TCA/acetone for protein extraction can preserve protein structure and function. These precipitating agents do not denature proteins, allowing analysis of proteins in their native or active form.
Simplified sample processing: Protein extraction with TCA/acetone is relatively simple and fast, providing a convenient approach to processing biological samples. This is especially advantageous when working with large numbers of samples or in situations where time is critical.
In summary, protein extraction with TCA/acetone is an important and versatile method that allows obtaining high quality proteins for various analyzes and studies. Its efficiency in removing contaminants, preserving protein structure and simplicity make it an essential technique in biomedical and biotechnological research.
Materials
Necessary materials:
1) Biological sample containing the proteins of interest
2) TCA (trichloroacetic acid) concentrate
3) Acetone 80% (cooled to -20°C)
4) Detergent (SDS 10%)
5) centrifuge
6) Microcentrifuge tubes
7) Ice
8) Tris-HCL 6.8 (tris(hydroxymethyl)aminomethane)
Prepare a 20% TCA solution by adding 200g of TCA in 800ml of distilled water. Be sure to do this in a well-ventilated area, wearing gloves and eye protection, as TCA is corrosive.
Add the biological sample (approximately 300 mg) to a 2 mL microcentrifuge tube.
Add a small amount of detergent (10% SDS) to the sample, if necessary, to break down cell membranes and facilitate protein release. The exact amount of detergent depends on the sample type and must be optimized experimentally.
Transfer the homogenized sample to a new microcentrifuge tube and centrifuge at high speed (eg 13,000 rpm) at 4°C for 20 minutes to remove cell debris and nuclei.
Discard the supernatant carefully, without disturbing the pellet formed at the bottom of the tube.
Add concentrated TCA (20% final) to the pellet containing the proteins, in a ratio of 4 parts of TCA to 1 part of the sample. For example, if you have 200 µl of sample, add 800 µl of TCA.
Incubate the sample on ice for 20-30 minutes to allow protein precipitation.
Centrifuge the sample at 4°C for 20 minutes at high speed (eg 13,000 rpm).
Carefully discard the supernatant and add 1 ml of 80% acetone (cooled to -20°C) to the pellet. Make sure the acetone completely covers the pellet.
Note: Wash the pellet with 80% acetone, 2x.
To do this, simply add 80% acetone and quickly stir using a vortex tube stirrer and centrifuge at 13,000 RPM for 5 min.
Incubate the sample on ice for 5 minutes to allow for further protein precipitation.
Centrifuge the sample at 4°C for 20 minutes at high speed (eg 13,000 rpm).
Carefully discard the supernatant and dry the protein pellet at room temperature.
The dried protein pellet can be resuspended in an extraction buffer (10% SDS with 6.8 tris-HCl) suitable for further analysis or apply 10 µL onto the SDS-PAGE electrophoresis gel.
Protocol references
Shiose, Lumi et al. Seleção de método de extração de proteínas de folhas de feijoeiro (Phaseolus vulgaris L.). 2019.
Wang, H., Joseph, J. A., & Quant, P. A. (1998). Development of a Simple and Rapid Protocol for the Isolation of Functional Proteins from Mammalian Cells. Biotechnology Progress, 14(3), 458-462.
Uyar, E., & Kilinc, E. (2014). Protein precipitation methods for proteomic analysis. Methods in Molecular Biology, 1072, 133-141.
Li, Y., Liang, Z., & Zhang, Z. (2014). Improved Protein Extraction and Protein Identification from Archival Formalin-fixed, Paraffin-embedded Human Aortas. Proteomics, 14(17-18), 1963-1970.
Clavreul, N., Ait-Belkacem, R., & Theret, N. (2017). Proteomic Approaches of Normal and Altered Protein Secretion in Human Cells: Towards the Definition of a Molecular Signature of Pathological Perturbations. Proteomes, 5(3), 20.
Liu, X., Chen, Z., Chua, H. N., & Jin, G. (2018). The Effect of Different Protein Extraction Methods on the Proteome Profile of the Immature Cotton Fiber. Frontiers in Plant Science, 9, 6.