Abstract LB-288: Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis

Abstract

Abstract Background: Formalin fixed paraffin embedded (FFPE) tissues remains the standard method for fixation and storage of tissues for clinical pathology use. Protein extraction from these tissues remains challenging due to the reduced quality and amount of extracted proteins. Despite multiple successful attempts, isolation of proteins from FFPE tissue sections necessitates routine use of xylene, a highly toxic organic solvent. We previously showed that proteins can be efficiently extracted with a novel technique that utilizes hot distilled water as a substitute for xylene with a quality adequate for western blot analysis. However, its major drawback is the need to use an entire or major part of the paraffin-embedded tissue block. To address these issues we developed a new xylene-free method for protein extraction from FFPE tissue sections of around 8 μm thickness. Methods: A total of 44 different types of FFPE tissues sections of 8 μm thickness were obtained from various archived FFPE specimens which include: 17 colorectal cancer (5-6 years), 7 breast cancer (3 years), 3 thyroid cancer (2 years), 4 ovarian cancer (2 years), 8 uterine cancer (1 year), and 5 prostate cancer (1 year). Deparaffinization was conducted by gentle treatment of each section with hot distilled water (≈90°C) for less than 10 seconds. Deparaffinized tissues were then placed in a cell lysis buffer and Laemli buffer and incubated at 100°C for 5-10 minutes. The extracted proteins were quantified using NanoDrop Spectrophotometer and evaluated using western blot analysis for the presence of AKT and beta-actin. Results: Using this method, a significant amount of proteins was successfully isolated with an average amount of 2.670 μg/μl ± 0.623 or 1068 μg/8 μm tissue section. Compared to the standard protein extraction method using xylene the amount of proteins extracted in this experiment is at least four times greater. Protein extracts were of good quality and efficiently analyzed by western blot experiment. Moreover, the isolated proteins showed a similar migration pattern compared to the positive control protein on SDS-PAGE but with better bands’ intensity and clarity. Protein kinase B (PKB/AKT) was successfully identified in all specimens, and beta-actin protein was resolved with an efficiency higher than 80%. Hence, this technique has enabled us to efficiently extract and detect selected proteins from archived samples for up to 6 years. Conclusion: We developed an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissues sections and adequate for western blot analysis. Hence, utilizing this technique can further aid in the identification of tissue protein biomarkers of various diseases, monitor cancer progression, and improve the diagnosis. It remains to be determined if this method of protein extraction form FFPE is adequate for use in proteomic analyses. Citation Format: Anthony Mansour, Noha Bejjani, Carole Dagher, Rajaa Chatila, Wissam H. Faour. Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-288. doi:10.1158/1538-7445.AM2015-LB-288