Deparaffinization and Protein Extraction from FFPE

Facilitate protein extraction from formalin-fixed paraffin-embedded (FFPE) tissue sections using an optimized combination of deparaffinization, solubilization, and sonication with a VialTweeter multi-tube ultrasonicator. This protocol supports downstream mass spectrometry–based proteomics and is compatible with SP3 cleanup and enzymatic digestion workflows.

This SOP is intended for laboratory personnel involved in proteomic analysis of FFPE tissue specimens using high-performance sonication. It is optimized for up to ten FFPE samples processed in parallel using the VialTweeter Multi-Tube Sonicator.

Protocol: Protein Extraction from FFPE Samples using the VialTweeter

Materials and Reagents

Reagents

• Xylene (histology grade)
• Ethanol (absolute 96%)
• Lysis buffer:

6 M Guanidine hydrochloride
50 mM Tris-HCl, pH 8.5
10 mM TCEP (Tris(2-carboxyethyl)phosphine)
40 mM CAA (2-Chloroacetamide)

• Protease inhibitor (optional; e.g., cOmplete™ mini EDTA-free)

Equipment

• VialTweeter Multi-Tube Ultrasonicator
• 1.5 mL or 2.0 mL low-binding microcentrifuge tubes
• Heat block or incubator (95°C and 80°C settings)
• Microcentrifuge
• Thermomixer (optional but recommended)

Sample Input

• 1–2 sections of 10 µm thick FFPE tissue per sample (i.e., per vial)

OR

• A total of ~100 µg tissue per sample (i.e., per vial)

Note: Use fresh blades for microtomy to minimize paraffin debris contamination.

Procedure

1. Deparaffinization
   1. Transfer FFPE sections into low-binding microcentrifuge tubes.
   2. Add 1 mL of xylene, vortex briefly.
   3. Incubate 10 minutes at room temperature.
   4. Centrifuge at 14,000 × g for 2 minutes; discard supernatant.
   5. Repeat xylene wash one more time (Steps 2–4).
   6. Wash pellet with 1 mL of 96% ethanol, vortex, then centrifuge at 14,000 × g for 2 minutes. Discard supernatant.
   7. Repeat ethanol wash once more (total of 2 ethanol washes).
   8. Air-dry pellet for 10 minutes at room temperature with open lids to evaporate residual ethanol.
2. Protein Extraction and Sonication
   1. Add 200 µL lysis buffer to each dry pellet.

      Note: Although the sonicator accommodates up to 1 mL, 200 µL is optimal for downstream processing.

   2. Mix by vortexing or gentle pipetting.
3. First Thermal Incubation
   1. Incubate the tubes at 95 °C for 30 min, with agitation at 400 rpm using a thermomixer or heat block.
   2. Let samples cool at room temperature for 5 min.
4. First Sonication with the VialTweeter
   1. Place the tubes into the VialTweeter.
   2. Set the VialTweeter UP200St to the values below and sonicate.
Sonicator Settings
• Set the Amplitude (A) to 100%
• Set the pulsation mode (C) to 100%
• Period Clock: On
• On Time: 60s
• Off Time: 30s
• Limit Value: 15 min (corresponds to 10 cycles)
(Calculation Note: (60 s On + 30 s Off) × 10 cycles = 900 s = 15 min)
5. Second Thermal Incubation
   Remove tubes and incubate again at 95 °C for 15 min, 400 rpm.
6. Second Sonication
   Repeat sonication on VialTweeter using the same settings (as above) for an additional 10 cycles (15 min total).
7. Clarification of Lysate
   1. Centrifuge the samples at 13,000 × g for 10 min at 23°C (room temperature).
   2. Carefully collect the supernatant into a new 2.0 mL Safe-lock Eppendorf tube. Avoid disturbing the pellet.
8. Downstream Processing
   The lysate is now ready for SP3 cleanup and enzymatic digestion.

Notes and Best Practices

1. Overheating risk during sonication is mitigated by programmed ON/OFF cycling.
2. Avoid vortexing post-sonication to prevent protein aggregation.

Waste Disposal and Safety

The table below gives you an indication of the approximate processing capacity of our lab-size ultrasonicators: