Abstract
Introduction: The chip-based protein separation using Agilent 2100 Bioanalyzer is a promising tool for proteomic analysis. However, it has not been defined if the above-mentioned device is suitable for analysis of formalin-fixed paraffin-embedded (FFPE) proteins extracts.<br>The aim of the study: Therefore we performed the analysis aimed at testing if extracts from FFPE tissues are suitable for proteins detection using Agilent 2100 Bioanalyzer.<br>Material and methods: FFPE tissues and cell cultures were used for experiments. Proteins were extracted from them using Qproteome FFPE Tissue Kit and RIPA buffer, respectively. For protein analysis Bioanalyzer Instrument, Western Blot and immunohistochemistry (IHC) were applied.<br>Results: In FFPE extracts, using Bioanalyzer we failed to detect β-actin. However, Western Blot analysis proved β-actin presence in them. One of possible explanations of this phenomenon might be lack of antibody-antigen interaction during immunoprecipitation preceding Bioanalyzer analysis. We suspected that, Extraction Buffer (from Qproteome FFPE Tissue Kit) or β-mercaptoethanol (both present in FFPE protein extracts) might be responsible for the above-mentioned blockade. Therefore, we applied IHC with detection of CD34 because, CD34 staining is robust against small methodological variations and this marker is always present in tumor tissue sections. Anti-CD34 antibody was diluted in Tris saline buffer with Extraction Buffer, β-mercaptoethanol or without. We did not observe CD34 immunopositivity only in the presence of Extraction Buffer.<br>Conclusion: Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer, because the Extraction Buffer from the kit prevents an interaction between antibody and antigen during immunoprecipitation procedure.
Published Version
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