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Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A

Prgomet, Z. LU ; Andersson, Tommy LU and Lindberg, P. LU (2017) In Biotechnic and Histochemistry 92(1). p.46-58
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Biotechnic and Histochemistry
volume
92
issue
1
pages
46 - 58
publisher
Informa Healthcare
external identifiers
  • scopus:85011586425
  • wos:000395756300006
ISSN
1052-0295
DOI
10.1080/10520295.2016.1255995
language
English
LU publication?
yes
id
e7e360ba-6443-4a01-9b35-c03f16b2e34b
date added to LUP
2017-02-20 09:53:00
date last changed
2018-01-07 11:51:01
@article{e7e360ba-6443-4a01-9b35-c03f16b2e34b,
  abstract     = {<p>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.</p>},
  author       = {Prgomet, Z. and Andersson, Tommy and Lindberg, P.},
  issn         = {1052-0295},
  language     = {eng},
  number       = {1},
  pages        = {46--58},
  publisher    = {Informa Healthcare},
  series       = {Biotechnic and Histochemistry},
  title        = {Optimization, validation, and identification of two reliable antibodies for immunodetection of WNT5A},
  url          = {http://dx.doi.org/10.1080/10520295.2016.1255995},
  volume       = {92},
  year         = {2017},
}