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/EBP beta expression during myogenesis. Though the antibody is monoclonal, and the immunogen used is highly specific to C/EBP beta, we identified an intense band at 23 kDa on western blot that did not correspond to any of the known isoforms of C/EBP beta, or family members predicted to cross-react. Absent in myoblast cells overexpressing C/EBP beta, the band was present when C/EBP beta was knocked down, confirming specificity for a protein other than C/EBP beta. 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/EBP beta 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/EBP beta isoforms.