Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A.
WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.
a Oral Pathology, Faculty of Odontology , Malmo University , Malmo , Sweden.|b Cell and Experimental Pathology, Department of Translational Medicine, Lund University , Clinical Research Centre, Skane University Hospital , Malmo , Sweden.
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Z
Prgomet
b Cell and Experimental Pathology, Department of Translational Medicine, Lund University , Clinical Research Centre, Skane University Hospital , Malmo , Sweden.
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T
Andersson
a Oral Pathology, Faculty of Odontology , Malmo University , Malmo , Sweden.
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P
Lindberg
D000906:Antibodies / Q000276:immunology*; D014408:Biomarkers, Tumor; D015153:Blotting, Western; D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D015203:Reproducibility of Results; D013194:Staining and Labeling; D000071818:Wnt-5a Protein / Q000737:chemistry / Q000276:immunology*
D000906:Antibodies; D014408:Biomarkers, Tumor; C085389:WNT5A protein, human; D000071818:Wnt-5a Protein
WNT5A; antibodies; immunohistochemistry; optimization; pre-absorption test; validation; wsestern blot analysis