Background: The two isoforms of the eukaryotic Elongation Factor 1A (eEF1A1 and eEF1A2), sustain theprogression/aggressiveness of cancer cells. Thus, they are considered promising therapeutic targets andprognostic markers. It follows that their precise quantification is of utmost relevance in research anddevelopment. The simultaneous quantification of A1 and A2 proteins in the cells helps the comprehen-sion of cancer biology mechanisms and response to drug treatments. However, the high homology atthe amino-acidic level (92%) can cause antibodies cross-reaction. Moreover, the commonly employedwestern blotting just gives semi-quantitative data and does not allow the detection of both protein tar-gets within the same cell. Thus, we developed an in cell western (ICW) technique to bypass the abovelimitations.Methods: Firstly, relevant antibodies cross-reaction was excluded by immunohistochemistry on normalpancreatic tissue; then eEF1A1-A2 protein levels were quantitated by ICW in prostate and colorectalcancer cell lines in 96 well plates under different conditions, which include: 1) drug treatment, 2) siRNAsilencing, 3) cell seeding density.Results: We show that: 1) eEF1A1-A2 levels vary depending on the cell type following drug treatment,2) ICW can accurately detect eEF1A1-A2 protein levels following siRNA silencing, 3) cell seeding densityinfluences eEF1A1-A2 levels, depending on cell type.Conclusions: ICW is a valuable tool to specifically determine the intracellular level of eEF1A1-A2 proteinsthus contributing to better define their role as potential therapeutic targets and prognostic markers inhuman tumors as well as for drug effects screening.
