Table 2.

Detailed description of the protocol and procedure.

Step by step protocol (overview)	A piece of tissue (about 1 mm3) is added to the extraction urea-containing buffer and glass beads. Buffer must cover the sample completely. Tissue is disrupted first by vortexing. To disintegrate the tissue further, perform sonication for 30 min, tube with sample must be kept on ice during procedure to avoid heating of the sample. Centrifugation at 13,000 g for 30 min on +4 °C is performed to clarify sample and to provide better compression of the pellet and better concentration of lipids in the top layer of the sample. After collection of the supernatant, concentration of the protein was determined with the use of a RC/DC kit. 80–100 μg of proteins is loaded on the IPG strip for 2D-GE.
Steps	Description	Reagents and tools	Troubleshoots
Evaluation of a sample	Clinical samples have to be evaluated by a pathologist. Quantity and quality of cellular, stromal and other histological features have to be evaluated.	Light microscope.	Selection of a non-appropriate sample will affect results of the experiment.
Extraction of proteins from the tissue	Tissue must be homogenized, proteins extracted and separated from the pellet. Sonication can be performed in steps, eg, 3 times of 10 min each. It is important that the sample is not heated upon extraction. Centrifugation can be repeated to improve separation from lipids and the pellet.	Urea buffer: 8 M urea, 2% (w\v) ChAPS, 50 mM DTT, 0.8% (v\v) ampholytes (pH range of ampholytes depends on used strips). Glass beads can be added in a proportion of 1/3 (beads/sample; v/v). Water-bath, sonicator, a centrifuge with cooling.	Particles, lipids and impurities in the sample lead to the distortions of protein separation during 2D-GE. During extraction procedure, it is important to control temperature. Especially during sonication and centrifugation. Urea may crystallize at low temperature (eg, +4 °C), while temperature higher than +20 °C may induce degradation processes in a sample. Shorter than 30 min centrifugation time may not be sufficient for efficient separation.
Evaluation of protein concentration	Optimal concentration of the protein required for achieving good quality of 2D gels. Usually, 80–100 μg of protein is enough to prepare one 2D maxi-gel (20 cm × 20 cm).	RC/DC kit or a similar kit.	Overload with proteins will make analysis of the separated proteins difficult due to very intense staining. If concentration is too low only few protein will be visualized in the 2D gels.