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Identification of Novel CK2 Kinase Substrates Using a Versatile Biochemical Approach
Published on: February 21, 2019
Potential misidentification of cyclooxygenase-2 by Western blot analysis and prevention through the inclusion of
Yen-Ting Liu1, Adel Kardosh, Janine Cooc
1Department of Molecular Microbiology and Immunology, University of Southern California, 2011 Zonal Ave., HMR-405, Los Angeles, CA 90089-9094, USA.
Abstract:
Cyclooxygenase (COX)-2 plays an important role in the development of cancer and has been recognized as a potential therapeutic target. Because nonsteroidal anti-inflammatory drugs (NSAIDs) are able to inhibit the activity of this enzyme, the potential efficacy of such drugs for purposes of cancer prevention or therapy is an area of intense research. Therefore, it is of critical importance to unequivocally determine the expression levels of COX-2 protein in tumor cells. In this regard, there are several conflicting reports in the literature where the same type of tumor cell lines were reported as COX-2 positive and as COX-2 negative. We found that during Western blot analysis of COX-2 positive and COX-2 negative cells, different antibodies to COX-2 protein are able to generate strong signals, which are false-positives and can be confused with COX-2. Thus, we believe that some of the conflicting reports on COX-2 expression in tumor cell lines could be the result of improper interpretation of the Western blot signals. Here, we present some of these pitfalls and suggest the inclusion of appropriate controls to unequivocally identify COX-2 protein levels.
Insights
Accurately measuring cyclooxygenase (COX)-2 protein is crucial for cancer research. This study identifies pitfalls in Western blot analysis, highlighting how false-positive signals can lead to conflicting COX-2 expression data in tumor cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cyclooxygenase (COX)-2 is implicated in cancer development and is a therapeutic target.
- Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit COX-2, prompting research into their cancer prevention and therapy potential.
- Accurate determination of COX-2 protein levels in tumor cells is critical for this research.
Purpose of the Study:
- To address conflicting reports on COX-2 expression in tumor cell lines.
- To identify potential sources of error in Western blot analysis for COX-2.
- To propose methods for unequivocally identifying COX-2 protein levels.
Main Methods:
- Western blot analysis was used to examine COX-2 protein expression.
- Comparison of results using different COX-2 antibodies.
- Inclusion of appropriate controls to validate results.
Main Results:
- Certain antibodies against COX-2 can produce strong signals that are false-positives.
- These false-positive signals can be mistaken for genuine COX-2 expression.
- Conflicting reports on COX-2 expression may stem from misinterpretation of Western blot data.
Conclusions:
- Improper interpretation of Western blot signals can lead to erroneous conclusions about COX-2 expression.
- The use of specific, validated antibodies and appropriate controls is essential.
- Standardizing Western blot protocols is necessary for reliable COX-2 detection in cancer research.
