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.
Highlights
We found that the GST-Extracellular signal-regulated kinase 8 (ERK8)(28aa) fusion protein was rapidly and highly expressed in E. coli as expected
We have recently shown that ERK8 can be activated by arsenic trioxide and activated ERK8 subsequently promotes the phosphorylation and degradation of IκBα, leading to the activation of NF-κB and lung cancer cell apoptosis [15]
The polypeptide antigen sequence we selected was located at the C-terminal region of ERK8, while most of commercial ERK8 antibodies were raised against the N-terminal region; which may be the reason of our home-made anti-ERK8 antibody’s high performance over commercial ERK8 antibodies
Summary
We report the design, expression, and purification of the recombinant ERK8 fusion protein, i.e., GST-ERK8(28aa), a designated 28 amino acid region of human ERK8 fused to GST and use it as an antigen to generate the anti-ERK8 polyclonal antibody. To develop an antibody recognizing ERK8 protein, we predicted the B-cell epitopes of human ERK8 using an online bioinformatics server Immune Epitope Database and Analysis Resource.
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