Conclusions
Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.
Methods
Using the example of urothelial carcinoma, FFPE tissue sections were subjected to various deparaffinization, protein extraction and antigen retrieval protocols and buffers as well as different extraction techniques. Performance was measured by protein concentration and western blot analysis of cellular compartment markers as well as liquid chromatography-coupled mass spectrometry (LC-MS).
Results
Commercially available extraction buffers showed reduced extraction of MPs and came at considerably increased costs. On-slide extraction did not improve PE whereas several other preanalytical steps could be simplified. Systematic variation of temperature and exposure duration demonstrated a quantitatively relevant corridor of optimal antigen retrieval. Conclusions: Preanalytical protein extraction can be optimized at various levels to improve unbiased protein extraction and to reduce time and costs.