Abstract
In most hospitals worldwide formalin-fixation and paraffin-embedding (FFPE) is the standard tissue fixation and storage method. The analysis and extraction of full-length and immunoreactive proteins from FFPE tissues was long believed to be impossible due to protein cross-linking during fixation. Nevertheless, in recent years, several research groups have successfully established protocols to extract proteins from FFPE tissues. For use in clinical in vitro diagnostics the extraction protocol should not be too complex and has to be compatible with downstream molecular analysis such as Western-blot, reverse phase protein array, or mass-spectrometry. Here we describe a protocol for protein extraction from FFPE tissues that fulfills these requirements.
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Notes
- 1.
For more details, see the QProteome FFPE Tissue Handbook, QIAGEN.
- 2.
100% isopropanol could also be used instead of ethanol.
- 3.
Other quantification methods can be used, such as the Bradford one (Bio-Rad Protein Assay Kit II, cat. No. 500-0002), the EZQ Protein Quantitation Kit (Molecular Probes Invitrogen, cat. No. R33200), or Quant-iT Protein Assay Kit (Molecular Probes Invitrogen, cat. No. Q33211).
- 4.
The paraffin blocks should be kept at −20°C or on dry ice in aluminum foil to obtain thin sections. Clean the microtome with xylene after each cutting.
- 5.
Alternatively, 100% isopropanol can be used.
- 6.
The volume of the extraction buffer should be adjusted according to the size of the tissue area. For an area of about 0.5 cm in diameter use 100 μl buffer.
- 7.
Be sure that collection tubes are properly sealed with a Collection Tube Sealing Clip before performing the next step.
- 8.
Other quantification methods can be used, such as the Bradford one (Bio-Rad Protein Assay Kit II, cat. No. 500-0002), the EZQ Protein Quantitation Kit (Molecular Probes Invitrogen, cat. No. R33200), or Quant-iT Protein Assay Kit (Molecular Probes Invitrogen, cat. No. Q33211).
- 9.
The paraffin blocks should be kept at −20°C or on dry ice in aluminum foil to obtain thin sections. Clean the microtome with xylene after each cutting.
- 10.
The cut sections should be stored at −20°C before performing the deparaffinization step if not immediately used.
- 11.
If the pellet detaches from the wall of the tube, repeat centrifugation step to enable removal of any residual ethanol. Do not disturb or remove the pellet.
- 12.
The volume of the extraction buffer should be adjusted according to the size of the tissue area. For an area of about 0.5 cm in diameter use 100 μl.
- 13.
Be sure that Collection Tubes are properly sealed with a Collection Tube Sealing Clip.
- 14.
Be sure that Collection Tube Sealing Clip has been removed before starting the centrifugation step.
- 15.
Due to the presence of detergents, the lysate should be diluted with the same volume of distilled water before performing the protein quantification; otherwise you should have some interference.
References
Becker KF, Schott C, Hipp S, Metzger V, Porschewski P, Beck R, Nahrig J, Becker I, Hofler H (2007) Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol 211(3):370–378
Wolff C, Schott C, Porschewski P, Reischauer B, Becker KF (2011) Successful protein extraction from over-fixed and long-term stored formalin-fixed tissues. PLoS One 6(1):e16353
Becker KF, Schott C, Becker I, Höfler H (2008) Guided protein extraction from formalin-fixed tissues for quantitative multiplex analysis avoids detrimental effects of histological stains. Proteomics Clin Appl 2(5):737–743
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Becker, KF., Schott, C. (2011). Protein Extraction from Formalin-Fixed Paraffin-Embedded Tissues. In: Stanta, G. (eds) Guidelines for Molecular Analysis in Archive Tissues. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17890-0_37
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DOI: https://doi.org/10.1007/978-3-642-17890-0_37
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