Qproteome FFPE Tissue Kit (20)

Protocol tips

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	To ensure maximum protein extraction, first, heat the homogenized tissues at 90°C for 20 min followed by heating (for 2 h for tissues in FFPE buffer) at 80°C.

Protocol tips
To ensure maximum protein extraction, first, heat the homogenized tissues at 90°C for 20 min followed by heating (for 2 h for tissues in FFPE buffer) at 80°C.

Publication protocol

Protein extraction from C-MFPE samples: C-MFPE samples (both scrapings and tissues) processed using automated Cellient® cell block system was sectioned into 5-7 um tissue curls. These were deparaffinized in xylene, washed in ethanol followed-by rehydration in phosphate buffered saline (PBS) and centrifuged at 14,000 g for 30 s. The resulting pellet was divided into three aliquots [Figure 2]. The aliquots were weighed and suspended in (1) Qproteome FFPE Tissue Kit (Qiagen Corp, CA, USA) or (2) Qproteome Mammalian Tissue kit (Qiagen Corp, CA, USA) or (3) Optimized CHAPS lysis buffer (30 mM Tris-Cl pH 7.5, 150 mM NaCl, 1% CHAPS, 0.03 mM dithiothreitol, protease inhibitor cocktail). The suspension was taken through 5-6 cycles of freeze-thaw to ensure maximum tissue lysis. The freezing was performed on dry ice for 5 min followed-by 10 min of thawing at 37°C. The freeze-thawed suspension was homogenized using TissueLyser LT (Qiagen Corp, CA, USA). To ensure maximum protein extraction, the homogenized tissues were first heated at 90°C for 20 min followed-by heating (for 2 h for tissues in FFPE buffer and 1 h for tissues in mammalian tissue buffer, as well as CHAPS buffer) at 80°C. The resultant lysate was centrifuged at full speed for 10 min and the supernatant was taken out for bicinchoninic acid (BCA) assay.

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