Novel Method for High Quality Protein Extraction from Adipose Tissues – Invent Biotechnologies Inc.
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Novel Method for High Quality Protein Extraction from Adipose Tissues

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Extracting total protein from adipose tissue can be challenging due to various factors. Adipose tissue consists of fat cells called adipocytes, surrounded by connective tissue and extracellular matrix. These components can interfere with protein extraction. Additionally, adipose tissue contains high levels of lipids, which can co-precipitate with proteins during extraction, resulting in low yields and poor-quality protein samples. The complexity of adipose tissue protein composition, which varies depending on the tissue's location and physiological state, further complicates the extraction process. Thus, optimizing protein extraction protocols for adipose tissue requires careful consideration of factors like buffer composition, homogenization method, and centrifugation parameters.

To overcome these challenges, several methods have been developed for extracting total protein from adipose tissue, including chemical lysis using RIPA buffer, mechanical homogenization, sonication, and organic solvent extraction. However, these methods often involve the use of large amounts of detergents, which can lead to protein loss and poor yield due to protein sequestration into micelles. Moreover, the presence of lipids in protein samples can significantly affect the band patterns observed in SDS-PAGE.

Zakharchenko et al. [1] conducted a study comparing the effect of different extraction buffers on the quality of extracted proteins from breast tissues. They found that regardless of the extraction buffer used, the protein bands appeared somewhat smeared in SDS-PAGE. The yield of proteins was generally low, and in some cases, a significant portion of proteins was lost (see figure below).

Lane assignment from left to right: Urea-containing extraction buffer, 1% Triton-X 100, RIPA buffer, 1% SDS followed by precipitation, and 1% SDS extraction (last lane)

In contrast to the above data, our spin-column-based kit (Cat#: AT-022) provides a simple and rapid solution for extracting total protein from adipose tissues with high yield and reproducible results. The kit utilizes a detergent-free buffer to prevent protein sequestration into micelles during homogenization of adipose tissue. The homogenate is briefly frozen as a water-oil emulsion and then passed through a filter cartridge. The filter efficiently separates the protein in the aqueous phase from the oil phase. After removing the lipids from the sample, the extracted total protein is solubilized. The following data demonstrate that the kit significantly improves both the yield and quality of protein extracts.

In a study investigating the relationship between PM2.5 exposure and the increased risk of obesity and type 2 diabetes [2], visceral white adipose tissue (vWAT), brown adipose tissue (BAT), and subcutaneous white adipose tissue (scWAT) were extracted from a murine model using the AT-022 kit. The extracted proteins were subjected to Western blotting analysis using different antibodies. As depicted in the Figure below, all protein bands appeared sharp and clean, with minimal background noise.

In another study conducted by Jahandideh, F. et al. [3], the mechanisms of egg white hydrolysate (EWH) for enhancing insulin sensitivity in insulin-resistant rats were investigated. Total protein from retroperitoneal fat and epididymal fat was extracted using the AT-022 kit. Semi-quantitative Western blotting was performed using the LICORE detection system to determine the relative expression of akt and phosphorylated akt. The findings indicated that EWH improves insulin sensitivity in insulin-resistant rats induced by a high-fat diet through akt activation. Accurate semi-quantification of protein bands requires clear and well-defined bands.

Oldoni F., et al. [4] explored the relationship between angiopoietin-like protein (A8) and triglyceride metabolism using mice lacking either liver-specific A8 (Ls-A8-/-) or adipose-specific A8 (As-A8-/-). Total protein was extracted from subcutaneous white adipose tissue (WAT-SQ) and brown adipose tissue (BAT) using the AT-022 kit. The extracted proteins were probed with anti-8A antibody and the loading control calnexin (CANX). Liver-specific A8 was found to be expressed in adipose tissues, while adipose-specific A8 was absent in both types of adipose tissues. The A8 bands and CANX bands appeared clear and well-defined, indicating the extraction of high-quality proteins.

 

Extracting total protein from adipose tissue can be a challenging process due to the complex composition of the tissue and its high lipid content. However, the use of the AT-022 kit enables quick and easy extraction of high-quality protein samples suitable for downstream analysis. While Western blotting is the most commonly used downstream application, the extracted proteins have also been utilized in ELISA [5]. The versatility of the AT-022 kit extends beyond laboratory animals such as mice and rats, as it has been successfully applied to various species including humans [6,7], pigs [8], cows [9], and cultured adipocytes [10].

References:

  1. Zakharchenko, O. et al. (2011) Optimized Protocol for Protein Extraction from the Breast Tissue that is Compatible with Two-Dimensional Gel Electrophoresis. Breast Cancer: Basic and Clinical Research 2011:5 37–42.
  2. Wang, N., Ma, Y., Liu, Z., Liu, L., Yang, K., Wei, Y., ... & Wen, D. (2019). Hydroxytyrosol prevents PM2. 5-induced adiposity and insulin resistance by restraining oxidative stress related NF-κB pathway and modulation of gut microbiota in a murine model. Free Radical Biology and Medicine, 141, 393-407. Zani, S. (2019).
  3. Jahandideh, F., de Campos Zani, S. C., Son, M., Proctor, S. D., Davidge, S. T., Chan, C. B., & Wu, J. (2019). Egg white hydrolysate enhances insulin sensitivity in high fat diet induced insulin resistant rats via AKT activation. British Journal of Nutrition, 1-25.
  4. Oldoni, F., Cheng, H., Banfi, S., Gusarova, V., Cohen, J. C., & Hobbs, H. H. (2020). ANGPTL8 has both endocrine and autocrine effects on substrate utilization. JCI insight, 5(17).
  5. Tu, J., Zhu, S., Li, B., Xu, G., Luo, X., Jiang, L., ... & Chen, C. (2020). Gegen Qinlian Decoction Coordinately Regulates PPARγ and PPARα to Improve Glucose and Lipid Homeostasis in Diabetic Rats and Insulin Resistance 3T3-L1 Adipocytes. Frontiers in Pharmacology, 11.
  6. Tu, J., Zhu, S., Li, B., Xu, G., Luo, X., Jiang, L., ... & Chen, C. (2020). Gegen Qinlian Decoction Coordinately Regulates PPARγ and PPARα to Improve Glucose and Lipid Homeostasis in Diabetic Rats and Insulin Resistance 3T3-L1 Adipocytes. Frontiers in Pharmacology, 11.
  7. Li, X., Zhang, X., Shen, Z., Chen, Z., Wang, H., & Zhang, X. (2022). GnRH receptor mediates lipid storage in female adipocytes via AMPK pathway. Int J Med Sci, 19(9), 1442-1450.
  8. Zheng, Q., Lin, J., Huang, J., Zhang, H., Zhang, R., Zhang, X., ... & Song, R. (2017). Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proceedings of the National Academy of Sciences, 201707853.
  9. Zhang, F., Li, D., Wu, Q., Sun, J., Guan, W., Hou, Y., ... & Wang, J. (2019). Prepartum body conditions affect insulin signaling pathways in postpartum adipose tissues in transition dairy cows. Journal of Animal Science and Biotechnology, 10(1), 38.
  10. Chen, T. X., Cheng, X. Y., Wang, Y., & Yin, W. (2018). Toosendanin inhibits adipogenesis by activating Wnt/β-catenin signaling. Scientific Reports, 8(1), 4626.Top of Form