Purification and characterization of a highly specific polyclonal antibody against human extracellular signal-regulated kinase 8 and its detection in lung cancer

PLoS One. 2017 Sep 13;12(9):e0184755. doi: 10.1371/journal.pone.0184755. eCollection 2017.

Abstract

Extracellular signal-regulated kinase 8 (ERK8), proposed as a novel potential therapeutic target for cancer, has been implicated in cell transformation, apoptosis, the protection of genomic integrity, and autophagy. To facilitate ERK8 research, a highly specific anti-ERK8 antibody is needed. In this article, we use the Immune Epitope Database and Analysis Resource online tool to predict B-cell epitopes of human ERK8 protein, and choose a 28 aa-peptide sequence to generate the GST-ERK8(28aa) fusion protein as the antigen for developing polyclonal antibody against ERK8. The specificity and sensitivity of anti-ERK8 antibody were robustly validated by immunoblotting, immunocytochemical and immunohistochemical analyses; and we found that both the endogenous and ectopically-expressed human ERK8 proteins can be recognized by our anti-ERK8 antibody. This suggested that our characterized anti-ERK8 antibody will be a valuable tool for the elucidation of the distribution of ERK8 at cellular and histological levels. Finally, our tissue array analysis also demonstrated that the ERK8 protein was localized in both the nucleus and cytoplasm of human lung cancers.

MeSH terms

  • Antibodies / chemistry*
  • Antibodies / isolation & purification
  • Antibody Specificity
  • Cell Line, Tumor
  • Cell Nucleus / metabolism
  • Cytoplasm / metabolism
  • Databases, Factual
  • Epitopes, B-Lymphocyte / chemistry*
  • Extracellular Signal-Regulated MAP Kinases / analysis
  • Extracellular Signal-Regulated MAP Kinases / immunology*
  • Humans
  • Immunohistochemistry
  • Lung Neoplasms / metabolism*
  • Software

Substances

  • Antibodies
  • Epitopes, B-Lymphocyte
  • Extracellular Signal-Regulated MAP Kinases
  • MAPK15 protein, human

Grant support

This work was supported by the grants from the National Natural Science Foundation of China (Nos. 31271445 and 31771582), the Science and Technology Planning Project of Guangdong Province of China (No. 2016A020215144), the Guangdong Natural Science Foundation of China (Nos. 2017A030313131 and S2012030006289), “Thousand, hundred, and ten" project of The Department of Education of Guangdong Province (No. 124), and the Department of Education, Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.