title: Protein multiplicity can lead to misconduct in western blotting and misinterpretation of immunohistochemical staining results, creating much conflicting data.
abstract: Western blotting (WB) and immunohistochemical staining (IHC) are common techniques for determining tissue protein expression. Both techniques require a primary antibody specific for the protein in question. WB data is band(s) on a membrane while IHC result is a staining on a tissue section. Most human genes are known to produce multiple protein isoforms; in agreement with that, multiple bands are often found on the WB membrane. However, a common but unspoken practice in WB is to cut away the extra band(s) and present for publication only the band of interest, which implies to the readers that only one form of protein is expressed and thus the data interpretation is straightforward. Similarly, few IHC studies discuss whether the antibody used is isoform-specific and whether the positive staining is derived from only one isoform. Currently, there is no reliable technique to determine the isoform-specificity of an antibody, especially for IHC. Therefore, cutting away extra band(s) on the membrane usually is a form of misconduct in WB, and a positive staining in IHC only indicates the presence of protein product(s) of the to-be-interrogated gene, and not necessarily the presence of the isoform of interest. We suggest that data of WB and IHC involving only one antibody should not be published and that relevant reports should discuss whether there may be protein multiplicity and whether the antibody used is isoform-specific. Hopefully, techniques will soon emerge that allow determination of not only the presence of protein products of genes but also the isoforms expressed.
authors.affiliation: Department of Cardiology Department, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, PR China. Electronic address: lxd@gmc.edu.cn.
authors.forename: Xingde
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Psychiatry, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, China.
authors.forename: Yiming
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, PR China. Electronic address: ypq1964@163.com.
authors.forename: Wenxiu
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Pathology, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, PR China; Department of Molecular Biology, Guizhou Medical University, Guiyang, Guizhou 550004, PR China. Electronic address: 1457658298@qq.com.
authors.forename: Zhizhong
authors.initials: Z
authors.lastname: Guan
authors.affiliation: Department of Molecular Biology, Guizhou Medical University, Guiyang, Guizhou 550004, PR China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Yu
authors.affiliation: Department of Pathology, Guizhou Medical University Hospital, Guiyang, Guizhou 550004, PR China; Department of Molecular Biology, Guizhou Medical University, Guiyang, Guizhou 550004, PR China. Electronic address: djliao@gmc.edu.cn.
authors.forename: D Joshua
authors.initials: DJ
authors.lastname: Liao
mesh_terms: D017398:Alternative Splicing; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D015982:Bias; D015153:Blotting, Western / Q000941:ethics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry / Q000941:ethics*; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D015871:Scientific Misconduct
chemical_list: D000906:Antibodies; C412476:DISC1 protein, human; D004268:DNA-Binding Proteins; D050506:Mutant Chimeric Proteins; D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C498305:TSNAX protein, human
keywords: Genome; Immunohistochemical staining; Protein isoform; Western blotting