Contaminating reactivity of a monoclonal CCAAT/Enhancer Binding Protein β antibody in differentiating myoblasts

BMC Res Notes. 2019 Oct 31;12(1):717. doi: 10.1186/s13104-019-4749-3.

Abstract

Objective: CCAAT/Enhancer Binding proteins (C/EBPs) are transcription factors involved in the regulation of a variety of cellular processes. We used the Abcam Recombinant Anti-C/EBP beta antibody (E299) to detect C/EBPβ expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBPβ, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBPβ, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBPβ, the band was present when C/EBPβ was knocked down, confirming specificity for a protein other than C/EBPβ. The objective of this work was to identify the contaminating reactivity.

Results: We performed immunoprecipitation followed by mass spectrometry to identified myosin light chain 4 (MYL4) as the unknown band, suggesting that the Abcam monoclonal antibody directed against C/EBPβ is not pure, but contains a contaminating antibody against MYL4. Caution should be used when working in cells lines that express MYL4 to not confound the detection of MYL4 with that of C/EBPβ isoforms.

Keywords: Antibody cross-reactivity; C/EBPβ; MYL4; Myoblast; Myogenesis.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / immunology*
  • Antibody Specificity / immunology
  • CCAAT-Enhancer-Binding Protein-beta / genetics
  • CCAAT-Enhancer-Binding Protein-beta / immunology*
  • CCAAT-Enhancer-Binding Protein-beta / metabolism
  • Cell Differentiation / genetics
  • Cell Differentiation / immunology*
  • Cell Line
  • Cross Reactions / immunology
  • Gene Expression Profiling
  • HEK293 Cells
  • Humans
  • Mice
  • Muscle Development / genetics
  • Muscle Development / immunology
  • Myoblasts / cytology
  • Myoblasts / immunology*
  • Myoblasts / metabolism
  • Myosin Light Chains / genetics
  • Myosin Light Chains / immunology
  • Myosin Light Chains / metabolism
  • Protein Isoforms / genetics
  • Protein Isoforms / immunology
  • Protein Isoforms / metabolism
  • Tandem Mass Spectrometry / methods

Substances

  • Antibodies, Monoclonal
  • CCAAT-Enhancer-Binding Protein-beta
  • CEBPB protein, human
  • MYL4 protein, human
  • Myosin Light Chains
  • Protein Isoforms